TY - JOUR A1 - Votteler, Miriam A1 - Carvajal Berrio, Daniel A. A1 - Pudlas, Marieke A1 - Walles, Heike A1 - Schenke-Layland, Katja T1 - Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy JF - Journal of Visual Expression N2 - Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10 Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization. KW - tissue engineering KW - label-free analysis KW - raman spectroscopy KW - bioengineering KW - living cells KW - extracellular matrix Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124569 VL - 63 IS - e3977 ER - TY - JOUR A1 - Moll, Corinna A1 - Reboredo, Jenny A1 - Schwarz, Thomas A1 - Appelt, Antje A1 - Schürlein, Sebastian A1 - Walles, Heike A1 - Nietzer, Sarah T1 - Tissue Engineering of a Human 3D in vitro Tumor Test System JF - Journal of Visualized Experiments N2 - Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system. Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns). Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow. In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model. KW - bioengineering KW - biomedical engineering KW - tissue engineering KW - biotechnology KW - cultured KW - tumor cells KW - cell culture KW - 3D in vitro models KW - bioreactor KW - dynamic culture conditions KW - tumor test system KW - primary cell isolation KW - BioVaSc KW - decellularization KW - equipment and supplies KW - cellular microenvironment KW - culture techniques KW - cell engineering KW - anatomy KW - physiology KW - molecular biology KW - cellular biology Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132277 UR - http://www.jove.com/video/50460 VL - 78 IS - e50460 ER - TY - JOUR A1 - Thibaudeau, Laure A1 - Taubenberger, Anna V. A1 - Holzapfel, Boris M. A1 - Quent, Verena M. A1 - Fuehrmann, Tobias A1 - Hesami, Parisa A1 - Brown, Toby D. A1 - Dalton, Paul D. A1 - Power, Carl A. A1 - Hollier, Brett G. A1 - Hutmacher, Dietmar W. T1 - A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone JF - Disease Models & Mechanisms N2 - The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact 'organ' bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo. KW - breast cancer KW - bone metastasis KW - humanized xenograft model KW - melt electrospinning KW - tissue engineering KW - osteotropism KW - in vivo KW - stem-cell niche KW - human prostate-cancer KW - morphogenetic protein KW - osteoprogenitor cells KW - endochondral ossification KW - mouse model KW - trabecular bone KW - calcium phosphate KW - skeletal metastases Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117466 VL - 7 IS - 2 ER - TY - JOUR A1 - Thibaudeau, Laure A1 - Taubenberger, Anna V. A1 - Theodoropoulos, Christina A1 - Holzapfel, Boris M. A1 - Ramuz, Olivier A1 - Straub, Melanie A1 - Hutmacher, Dietmar W. T1 - New mechanistic insights of integrin β1 in breast cancer bone colonization JF - Oncotarget N2 - Bone metastasis is a frequent and life-threatening complication of breast cancer. The molecular mechanisms supporting the establishment of breast cancer cells in the skeleton are still not fully understood, which may be attributed to the lack of suitable models that interrogate interactions between human breast cancer cells and the bone microenvironment. Although it is well-known that integrins mediate adhesion of malignant cells to bone extracellular matrix, their role during bone colonization remains unclear. Here, the role of β1 integrins in bone colonization was investigated using tissue-engineered humanized in vitro and in vivo bone models. In vitro, bone-metastatic breast cancer cells with suppressed integrin β1 expression showed reduced attachment, spreading, and migration within human bone matrix compared to control cells. Cell proliferation in vitro was not affected by β1 integrin knockdown, yet tumor growth in vivo within humanized bone microenvironments was significantly inhibited upon β1 integrin suppression, as revealed by quantitative in/ex vivo fluorescence imaging and histological analysis. Tumor cells invaded bone marrow spaces in the humanized bone and formed osteolytic lesions; osteoclastic bone resorption was, however, not reduced by β1 integrin knockdown. Taken together, we demonstrate that β1 integrins have a pivotal role in bone colonization using unique tissue-engineered humanized bone models. KW - tissue engineering KW - bone colonization KW - breast cancer KW - β1 integrin KW - humanized bone models Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175432 VL - 6 IS - 1 ER - TY - JOUR A1 - Hinderer, Svenja A1 - Shen, Nian A1 - Ringuette, Léa-Jeanne A1 - Hansmann, Jan A1 - Reinhardt, Dieter P A1 - Brucker, Sara Y A1 - Davis, Elaine C A1 - Schenke-Layland, Katja T1 - In vitro elastogenesis: instructing human vascular smooth muscle cells to generate an elastic fiber-containing extracellular matrix scaffold JF - Biomedical Materials N2 - Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased elastin expression followed by elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM. KW - elastin KW - elastic fibers KW - electrospinning KW - tissue engineering KW - regenerative medicine KW - heart valve KW - cardiovascular Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254074 VL - 10 IS - 3 ER - TY - JOUR A1 - Suliman, Salwa A1 - Sun, Yang A1 - Pedersen, Torbjorn O. A1 - Xue, Ying A1 - Nickel, Joachim A1 - Waag, Thilo A1 - Finne-Wistrand, Anna A1 - Steinmüller-Nethl, Doris A1 - Krueger, Anke A1 - Costea, Daniela E. A1 - Mustafa, Kamal T1 - In vivo host response and degradation of copolymer scaffolds functionalized with nanodiamonds and bone morphogenetic protein 2 JF - Advanced Healthcare Materials N2 - The aim is to evaluate the effect of modifying poly[(L-lactide)-co-(epsilon-caprolactone)] scaffolds (PLCL) with nanodiamonds (nDP) or with nDP+physisorbed BMP-2 (nDP+BMP-2) on in vivo host tissue response and degradation. The scaffolds are implanted subcutaneously in Balb/c mice and retrieved after 1, 8, and 27 weeks. Molecular weight analysis shows that modified scaffolds degrade faster than the unmodified. Gene analysis at week 1 shows highest expression of proinflammatory markers around nDP scaffolds; although the presence of inflammatory cells and foreign body giant cells is more prominent around the PLCL. Tissue regeneration markers are highly expressed in the nDP+BMP-2 scaffolds at week 8. A fibrous capsule is detectable by week 8, thinnest around nDP scaffolds and at week 27 thickest around PLCL scaffolds. mRNA levels of ALP, COL1 alpha 2, and ANGPT1 are signifi cantly upregulating in the nDP+BMP-2 scaffolds at week 1 with ectopic bone seen at week 8. Even when almost 90% of the scaffold is degraded at week 27, nDP are observable at implantation areas without adverse effects. In conclusion, modifying PLCL scaffolds with nDP does not aggravate the host response and physisorbed BMP-2 delivery attenuates infl ammation while lowering the dose of BMP-2 to a relatively safe and economical level. KW - inflammatory response KW - biocompatibility KW - BMP-2 delivery KW - inflammation KW - tissue engineering Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189764 VL - 5 IS - 6 ER - TY - JOUR A1 - Groeber, Florian A1 - Engelhardt, Lisa A1 - Lange, Julia A1 - Kurdyn, Szymon A1 - Schmid, Freia F. A1 - Rücker, Christoph A1 - Mielke, Stephan A1 - Walles, Heike A1 - Hansmann, Jan T1 - A First Vascularized Skin Equivalent as an Alternative to Animal Experimentation JF - ALTEX - Alternatives to Animal Experimentation N2 - Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin & eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research. KW - alternative to animal testing KW - skin equivalents KW - tissue engineering KW - vascularization Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164438 VL - 33 IS - 4 ER - TY - JOUR A1 - Lotz, C. A1 - Kiesewetter, L. A1 - Schmid, F. F. A1 - Hansmann, J. A1 - Walles, H. A1 - Groeber-Becker, F. T1 - Replacing the Draize eye test: impedance spectroscopy as a 3R method to discriminate between all GHS categories for eye irritation JF - Scientific Reports N2 - Highly invasive animal based test procedures for risk assessment such as the Draize eye test are under increasing criticism due to poor transferability for the human organism and animal-welfare concerns. However, besides all efforts, the Draize eye test is still not completely replaced by alternative animal-free methods. To develop an in vitro test to identify all categories of eye irritation, we combined organotypic cornea models based on primary human cells with an electrical readout system that measures the impedance of the test models. First, we showed that employing a primary human cornea epithelial cell based model is advantageous in native marker expression to the primary human epidermal keratinocytes derived models. Secondly, by employing a non-destructive measuring system based on impedance spectroscopy, we could increase the sensitivity of the test system. Thereby, all globally harmonized systems categories of eye irritation could be identified by repeated measurements over a period of 7 days. Based on a novel prediction model we achieved an accuracy of 78% with a reproducibility of 88.9% to determine all three categories of eye irritation in one single test. This could pave the way according to the 3R principle to replace the Draize eye test. KW - biological models KW - electrical and electronic engineering KW - regenerative medicine KW - tissue engineering KW - toxicology Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177492 VL - 8 IS - 15049 ER - TY - JOUR A1 - Bothe, Friederike A1 - Deubel, Anne-Kathrin A1 - Hesse, Eliane A1 - Lotz, Benedict A1 - Groll, Jürgen A1 - Werner, Carsten A1 - Richter, Wiltrud A1 - Hagmann, Sebastien T1 - Treatment of focal cartilage defects in minipigs with zonal chondrocyte/mesenchymal progenitor cell constructs JF - International Journal of Molecular Sciences N2 - Despite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (µCT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O’Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage. KW - cartilage repair KW - osteochondral defect KW - tissue engineering KW - starPEG hydrogel KW - chondrocyte KW - MSC KW - zonal construct KW - minipig Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285118 SN - 1422-0067 VL - 20 IS - 3 ER - TY - JOUR A1 - Schmalzl, Jonas A1 - Plumhoff, Piet A1 - Gilbert, Fabian A1 - Gohlke, Frank A1 - Konrads, Christian A1 - Brunner, Ulrich A1 - Jakob, Franz A1 - Ebert, Regina A1 - Steinert, Andre F. T1 - Tendon-derived stem cells from the long head of the biceps tendon JF - Bone & Joint Research N2 - Objectives The long head of the biceps (LHB) is often resected in shoulder surgery and could therefore serve as a cell source for tissue engineering approaches in the shoulder. However, whether it represents a suitable cell source for regenerative approaches, both in the inflamed and non-inflamed states, remains unclear. In the present study, inflamed and native human LHBs were comparatively characterized for features of regeneration. Methods In total, 22 resected LHB tendons were classified into inflamed samples (n = 11) and non-inflamed samples (n = 11). Proliferation potential and specific marker gene expression of primary LHB-derived cell cultures were analyzed. Multipotentiality, including osteogenic, adipogenic, chondrogenic, and tenogenic differentiation potential of both groups were compared under respective lineage-specific culture conditions. Results Inflammation does not seem to affect the proliferation rate of the isolated tendon-derived stem cells (TDSCs) and the tenogenic marker gene expression. Cells from both groups showed an equivalent osteogenic, adipogenic, chondrogenic and tenogenic differentiation potential in histology and real-time polymerase chain reaction (RT-PCR) analysis. Conclusion These results suggest that the LHB tendon might be a suitable cell source for regenerative approaches, both in inflamed and non-inflamed states. The LHB with and without tendinitis has been characterized as a novel source of TDSCs, which might facilitate treatment of degeneration and induction of regeneration in shoulder surgery. KW - biceps tendon KW - tendon-derived stem cell KW - mesenchymal stem cell KW - tissue engineering KW - shoulder Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200370 VL - 8 IS - 9 ER -