Bioprinting of vascularized bone tissue equivalents

Bone tissue is one of the most frequently transplanted tissues. Since procedures like the transplantation of autologous bone bear risks, though, regenerative medicine and tissue engineering reach to face those problems by engineering bone substitutes by using suitable materials and living cells. A crucial factor is the vascularization of the constructed tissue to ensure supply of the included cells with nutrients and oxygen. For the fabrication of such bone tissue equivalents, evolving manufacturing techniques like bioprinting can be used to construct geometrically defined three-dimensional structures. Please click Additional Files below to see the full abstract

3D biodegradable scaffolds of polycaprolactone with silicate-containing hydroxyapatite microparticles for bone tissue engineering: high-resolution tomography and in vitro study

To date, special interest has been paid to composite scaffolds based on polymers enriched with hydroxyapatite (HA). However, the role of HA containing different trace elements such as silicate in the structure of a polymer scaffold has not yet been fully explored. Here, we report the potential use of silicate-containing hydroxyapatite (SiHA) microparticles and microparticle aggregates in the predominant range from 2.23 to 12.40 µm in combination with polycaprolactone (PCL) as a hybrid scaffold with randomly oriented and well-aligned microfibers for regeneration of bone tissue. Chemical and mechanical properties of the developed 3D scaffolds were investigated with XRD, FTIR, EDX and tensile testing. Furthermore, the internal structure and surface morphology of the scaffolds were analyzed using synchrotron X-ray µCT and SEM. Upon culturing human mesenchymal stem cells (hMSC) on PCL-SiHA scaffolds, we found that both SiHA inclusion and microfiber orientation affected cell adhesion. The best hMSCs viability was revealed at 10 day for the PCL-SiHA scaffolds with well-aligned structure (~82%). It is expected that novel hybrid scaffolds of PCL will improve tissue ingrowth in vivo due to hydrophilic SiHA microparticles in combination with randomly oriented and well-aligned PCL microfibers, which mimic the structure of extracellular matrix of bone tissue

Author Correction: 3D biodegradable scaffolds of polycaprolactone with silicate-containing hydroxyapatite microparticles for bone tissue engineering: high-resolution tomography and in vitro study

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper

Electrospun poly(d/l-lactide-co-l-lactide) hybrid matrix: a novel scaffold material for soft tissue engineering

Electrospinning is a long-known polymer processing technique that has received more interest and attention in recent years due to its versatility and potential use in the field of biomedical research. The fabrication of three-dimensional (3D) electrospun matrices for drug delivery and tissue engineering is of particular interest. In the present study, we identified optimal conditions to generate novel electrospun polymeric scaffolds composed of poly-d/l-lactide and poly-l-lactide in the ratio 50:50. Scanning electron microscopic analyses revealed that the generated poly(d/l-lactide-co-l-lactide) electrospun hybrid microfibers possessed a unique porous high surface area mimicking native extracellular matrix (ECM). To assess cytocompatibility, we isolated dermal fibroblasts from human skin biopsies. After 5 days of in vitro culture, the fibroblasts adhered, migrated and proliferated on the newly created 3D scaffolds. Our data demonstrate the applicability of electrospun poly(d/l-lactide-co-l-lactide) scaffolds to serve as substrates for regenerative medicine applications with special focus on skin tissue engineering

Genome-wide identification and phenotypic characterization of seizure-associated copy number variations in 741,075 individuals

Copy number variants (CNV) are established risk factors for neurodevelopmental disorders with seizures or epilepsy. With the hypothesis that seizure disorders share genetic risk factors, we pooled CNV data from 10,590 individuals with seizure disorders, 16,109 individuals with clinically validated epilepsy, and 492,324 population controls and identified 25 genome-wide significant loci, 22 of which are novel for seizure disorders, such as deletions at 1p36.33, 1q44, 2p21-p16.3, 3q29, 8p23.3-p23.2, 9p24.3, 10q26.3, 15q11.2, 15q12-q13.1, 16p12.2, 17q21.31, duplications at 2q13, 9q34.3, 16p13.3, 17q12, 19p13.3, 20q13.33, and reciprocal CNVs at 16p11.2, and 22q11.21. Using genetic data from additional 248,751 individuals with 23 neuropsychiatric phenotypes, we explored the pleiotropy of these 25 loci. Finally, in a subset of individuals with epilepsy and detailed clinical data available, we performed phenome-wide association analyses between individual CNVs and clinical annotations categorized through the Human Phenotype Ontology (HPO). For six CNVs, we identified 19 significant associations with specific HPO terms and generated, for all CNVs, phenotype signatures across 17 clinical categories relevant for epileptologists. This is the most comprehensive investigation of CNVs in epilepsy and related seizure disorders, with potential implications for clinical practice

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Induction of morphological and physiological reactions of primary skin cells by bio-inspired nano- and microstructured substrates

Im Forschungsgebiet Biomaterialien ist die Entwicklung und Optimierung von Materialien für biologische Anwendungen wie beispielsweise Implantate und Prothesen von zentraler Bedeutung. Gegenwärtig liegt der Fokus auf bioinspirierten und biomimetischen Strategien, wobei die Kenntnis spezifischer Zell-Material-Wechselwirkungen im Mittelpunkt steht. Dieses Wissen ist auch für das Tissue Engineering essentiell, um Zellen funktionell zu kultivieren oder Stammzellnischen nachzuahmen. In dieser Arbeit wurden bioinspirierte Oberflächen hergestellt, die angelehnt an Proteine der extrazellulären Matrix globulär oder fibrillär strukturiert waren. Die Funktionalisierung der Substrate erfolgte mit Amino- oder Carboxygruppen. Mit Hilfe von REM, AFM, ESCA, Ellipsometrie und Kontaktwinkelmessungen ließen sich die erzeugten, modifizierten Substrate charakterisieren. MALDI-TOF MS diente zur Evaluierung der Proteinadsorption, die einen entscheidenden Einfluss auf zelluläre Reaktionen haben kann. Nach erfolgreicher Herstellung und Charakterisierung der Substrate wurden primäre humane Keratinozyten und Fibroblasten auf den Oberflächen kultiviert. Es zeigten sich deutliche und unterschiedliche Reaktionen der Zel¬len in Bezug auf ihre Morphologie und Physiologie. Keratinozyten verfügten auf rauen globulären und fibrillären Substraten über langgestreckte Zellkörper, die cha¬rakteristisch für migrierende Zellen sind. Fibroblasten richteten sich bei den rauen fibrillären Substraten parallel zu den Rillen aus. Diese Resultate verdeutlichen den Einfluss von bioinspirierten Geometrien, da mit der erzeugten fibrillären Strukturierung große Kollagen-Fibrillen nachgeahmt wurden, an die sich Fibroblasten in vivo anordnen. Fokalkontakte, die für die feste Verankerung adhärenter Zellen wichtig sind, waren auf allen Oberflächen nachzuweisen. Eine deutlich höhere Viabilität und Proliferation von Keratinozyten auf amino-modifizierten Substraten ergaben die WST-Messungen. Die Ausbildung von Zell-Zell Kontakten wurde bei keinem Zelltyp durch die Strukturierung und Funktionalisierung beeinträchtigt. Bei der Evaluierung des Differenzierungszustandes primärer Keratinozyten kamen auf allen Oberflächen über 95% früh differenzierte, Cytokeratin 14 exprimierende Zellen vor. Der als epidermaler Stammzellmarker diskutierte Transkriptionsfaktor p63 war in einzelnen Zellen auf allen Substraten nachzuweisen. Tendenziell schienen besonders Keratinozyten auf carboxy-modifizierten Oberflächen und am Rand von Kolonien diesen Marker zu besitzen, wohingegen hoch proliferative Zellen im Zentrum der Kolonien vorlagen. Eine Bestimmung der Funktio¬nalität von Fibroblasten erfolgte über den Nachweis der Kollagen Typ I Produktion. Die Syn¬these dieses Proteins war auf strukturierten und funktionalisierten Substraten nicht eingeschränkt. Bei REM-Aufnahmen zeigten sich Unterschiede in der Ausbildung von sensorischen Aktin-Ausstülpungen, den Filopodien. Kera¬tinozyten auf rauen Oberflächen bildeten deutlich weniger dieser Strukturen aus. Diese Arbeit liefert neue Erkenntnisse bezüglich zelltypspezifischer Material-Wechselwirkungen. Neben der Herstellung, Funktionalisierung und Charakterisierung von bioinspirierten Substraten konnten morphologische und physiologische Reaktionen primärer humaner Keratinozyten und Fibroblasten induziert werden.Research in biomaterial sciences focus on the development and optimization of materials for biological applications, e.g. implants and prostheses. Main topics are bioinspired or biomimetic strategies. Knowledge of specific cell-material interaction is crucially important, especially for the cultivation of functional cells and the in vitro imitation of stem cell niches in tissue engineering. Within the scope of this dissertation bio-inspired globular or fibrillar structured substrates were generated, modelled after extracellular matrix proteins. Surfaces were functionalized with amino- or carboxy groups. The produced modified substrates were evaluated by SEM, AFM, XPS, ellipsometry and contact angle measurements. For characterizing the protein adsorption, which is essential for cell adhesion, MALDI-TOF MS was performed. After their successful generation and characterization, pri¬mary human keratinocytes and fibroblasts were cultivated on the modified substrates. Different morphological and physiological reactions of keratinocytes and fibroblasts could be determined. On rough globular or fibrillar substrates the cell shape of keratinocytes was elongated, which is characteristic for migrating cells. Fibroblasts strongly aligned to rough fibrillar structures, which had similar dimensions to collagen fibrils in vivo. This result underlines the impact of bio-inspired geometries and dimensions in biomaterial sciences. The formation of focal adhesions, which is necessary for strong attachment to the surface, could be observed for adherent cells on all substrates. Based on WST-measurements a higher viability and proliferation were determined for keratinocytes cultured on amino-modified surfaces. Functionalization and structuring of the material interfaces did not have any negative effects on the formation of cell-cell contacts. Regarding the differentiation of keratino-cytes, more than 95% of the adherent cells on all substrates were very early differentiated keratinocytes, which express cytokeratin 14. P63 is discussed as an epidermal stem cell marker and could be detected in several keratinocytes on all sub-strates. While more p63 positive cells seemed to be at carboxy-modified substrates and at the edge of the colonies, highly proliferative keratinocytes were located in the centre of the colonies. Functionality of fibroblasts was verified by staining of collagen type I production. Fibroblasts showed no change in the protein syntheses on structured and functionalized surfaces. With SEM differences in the development of sensory actin-structures, so called filopodia were found. Keratinocytes on rough sub-strates had notably less of these structures than adherent cells on planar surfaces. The results give new insights into cell-material interactions of selected primary human skin cells. Bioinspired substrates were generated, functionalized and characterized. With these modified surfaces morphological and physiological reactions of keratinocytes and fibroblasts could be induce

Comparison of osteoclastogenesis and resorption activity of human osteoclasts on tissue culture polystyrene and on natural extracellular bone matrix in 2D and 3D

Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (αVβ3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts