TY - JOUR A1 - Kleinhans, Claudia A1 - Schmid, Freia A1 - Schmid, Franziska A1 - Kluger, Petra T1 - Comparison of osteoclastogenesis and resorption activity of human osteoclasts on tissue culture polystyrene and on natural extracellular bone matrix in 2D and 3D JF - Journal of biotechnology N2 - 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. KW - osteoclastogenesis KW - human monocytes KW - osteoclasts KW - cell–material interactions KW - bone resorption Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bsz:rt2-opus4-5287 SN - 0168-1656 VL - 205 SP - 101 EP - 110 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Kleinhans, Claudia A1 - Vacun, Gabriele A1 - Surmenev, Roman A1 - Surmeneva, Maria A1 - Kluger, Petra T1 - Testing the in vitro performance of hydroxyapatite coated magnesium (AZ91D) and titanium concerning cell adhesion and osteogenic differentiation JF - BioNanoMaterials N2 - In the current study the in vitro outcome of a degradable magnesium alloy (AZ91D) and standard titanium modified by nanostructured-hydroxyapatite (n-HA) coatings concerning cell adhesion and osteogenic differentiation was investigated by direct cell culture. The n-HA modification was prepared via radio-frequency magnetron sputtering deposition and proven by field emission scanning electron microscopy and X-ray powder diffraction patterns revealing a homogenous surface coating. Human mesenchymal stem cell (hMSCs) adhesion was examined after one and 14 days displaying an enhanced initial cell adhesion on the n-HA modified samples. The osteogenic lineage commitment of the cells was determined by alkaline phosphatase (ALP) quantification. On day one n-HA coated AZ91D exhibited a comparable ALP expression to standard tissue culture polystyrene samples. However, after 14 days solely little DNA and ALP amounts were measurable on n-HA coated AZ91D due to the lack of adherent cells. Titanium displayed excellent cell adhesion properties and ALP was detectable after 14 days. An increased pH of the culture was measured for AZ91D as well as for n-HA coated AZ91D. We conclude that n-HA modification improves initial cell attachment on AZ91D within the first 24 h. However, the effect does not ersist for 14 days in in vitro conditions. KW - cell adhesion KW - direct cell test KW - human mesenchymal stem cells KW - in vitro testing KW - magnesium alloys KW - nanostructured-hydroxyapatite coating KW - osteoinductivity Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bsz:rt2-opus4-5290 SN - 2196-0651 VL - 16 IS - 1 SP - 41 EP - 50 PB - De Gruyter CY - Berlin, Boston ER - TY - JOUR A1 - Kleinhans, Claudia A1 - Mohan, Ramkumar A1 - Vacun, Gabriele A1 - Schwarz, Thomas A1 - Haller, Barbara A1 - Sun, Yang A1 - Kahlig, Alexander A1 - Kluger, Petra A1 - Finne-Wistrand, Anna A1 - Walles, Heike A1 - Hansmann, Jan T1 - A perfusion bioreactor system efficiently generates cell-loaded bone substitute materials for addressing critical size bone defects JF - Biotechnology journal N2 - Critical size bone defects and non-union fractions are still challenging to treat. Cell-loaded bone substitutes have shown improved bone ingrowth and bone formation. However, a lack of methods for homogenously colonizing scaffolds limits the maximum volume of bone grafts. Additionally, therapy robustness is impaired by heterogeneous cell populations after graft generation. Our aim was to establish a technology for generating grafts with a size of 10.5 mm in diameter and 25 mm of height, and thus for grafts suited for treatment of critical size bone defects. Therefore, a novel tailor-made bioreactor system was developed, allowing standardized flow conditions in a porous poly(L-lactide co-caprolactone) material. Scaffolds were seeded with primary human mesenchymal stem cells derived from four different donors. In contrast to static experimental conditions, homogenous cell distributions were accomplished under dynamic culture. Additionally, culture in the bioreactor system allowed the induction of osteogenic lineage commitment after one week of culture without addition of soluble factors. This was demonstrated by quantitative analysis of calcification and gene expression markers related to osteogenic lineage. In conclusion, the novel bioreactor technology allows efficient and standardized conditions for generating bone substitutes that are suitable for the treatment of critical size defects in humans. KW - bone substitute KW - critical size defect KW - perfusion bioreactor system KW - poly(LLA-co-CL) scaffold KW - tissue engineering Y1 - 2015 U6 - http://dx.doi.org/10.1002/biot.201400813 SN - 1860-6768 VL - 10 IS - 11 SP - 1727 EP - 1738 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Surmeneva, Maria A1 - Kleinhans, Claudia A1 - Vacun, Gabriele A1 - Kluger, Petra A1 - Schönhaar, Veronika A1 - Müller, Michaela A1 - Hein, Sebastian Boris A1 - Wittmar, Alexandra A1 - Ulbricht, Mathias A1 - Prymak, Oleg A1 - Oehr, Christian A1 - Surmenev, Oleg T1 - Nano-hydroxyapatite-coated metal-ceramic composite ofiron-tricalcium Phosphate : improving the surface wettability,adhesion and proliferation of mesenchymal stem cells in vitro JF - Colloids and surfaces / B : biointerfaces N2 - Thin radio-frequency magnetron sputter deposited nano-hydroxyapatite (HA) films were prepared on the surface of a Fe-tricalcium phosphate (Fe-TCP) bioceramic composite, which was obtained using a conventional powder injection moulding technique. The obtained nano-hydroxyapatite coated Fe-TCP biocomposites (nano HA-Fe-TCP) were studied with respect to their chemical and phase composition, surface morphology, water contact angle, surface free energy and hysteresis. The deposition process resulted in a homogeneous, single-phase HA coating. The ability of the surface to support adhesion and the proliferation of human mesenchymal stem cells (hMSCs) was studied using biological short-term tests in vitro. The surface of the uncoated Fe-TCP bioceramic composite showed an initial cell attachment after 24 h of seeding, but adhesion, proliferation and growth did not persist during 14 days of culture.However, the HA-Fe-TCP surfaces allowed cell adhesion, and proliferation during 14 days. The deposition of the nano-HA films on the Fe-TCP surface resulted in higher surface energy, improved hydrophilicity and biocompatibility compared with the surface of the uncoated Fe-TCP. Furthermore, it is suggested that an increase in the polar component of the surface energy was responsible for the enhanced cell adhesion and proliferation in the case of the nano-HA Fe-TCP biocomposites. KW - bioceramic composite KW - bioresorbable alloy KW - hydroxyapatite coating KW - RF magnetron sputtering KW - cell adhesion Y1 - 2015 U6 - http://dx.doi.org/10.1016/j.colsurfb.2015.07.057 SN - 0927-7765 VL - 135 SP - 386 EP - 393 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Huber, Birgit A1 - Volz, Ann-Cathrin A1 - Kluger, Petra T1 - How do culture media influence in vitro perivascular cell behavior? JF - Cell biology international N2 - Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146 cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146 cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146 cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146⁺ perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries. KW - actin KW - CD146 KW - pericyte culture KW - NG2 KW - myosin Y1 - 2015 U6 - http://dx.doi.org/10.1002/cbin.10515 VL - 39 IS - 12 SP - 1395 EP - 1407 PB - Wiley-Blackwell CY - Hoboken, NJ ER - TY - JOUR A1 - Huber, Birgit A1 - Volz, Ann-Cathrin A1 - Kluger, Petra T1 - Understanding the effects of mature adipocytes and endothelial cells on fatty acid metabolism and vascular tone in physiological fatty tissue for vascularized adipose tissue engineering JF - Cell & tissue research N2 - Engineering of large vascularized adipose tissue constructs is still a challenge for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Communication between mature adipocytes and endothelial cells is important for homeostasis and the maintenance of adipose tissue mass but, to date, is mainly neglected in tissue engineering strategies. Thus, new coculture strategies are needed to integrate adipocytes and endothelial cells successfully into a functional construct. This review focuses on the cross-talk of mature adipocytes and endothelial cells and considers their influence on fatty acid metabolism and vascular tone. In addition, the properties and challenges with regard to these two cell types for vascularized tissue engineering are highlighted. KW - mature adipocytes KW - endothelial cells KW - co-culture KW - vascularized fatty tissue engineering KW - cross-talk Y1 - 2015 U6 - http://dx.doi.org/10.1007/s00441-015-2274-9 SN - 0302-766X VL - 362 IS - 2 SP - 269 EP - 279 PB - Springer CY - Berlin, Heidelberg ER - TY - JOUR A1 - Thude, Sibylle A1 - Kluger, Petra A1 - Schenke-Layland, Katja T1 - In vitro-Hauttestsysteme zur Untersuchung lichtassoziierter Hautschädigung JF - Biospektrum N2 - Sunlight has various effects on human health. Several important metabolic processes are only enabled by sunlight. But longtime sun bathing and extended outdoor activities can cause skin irritation, inflammation or even skin cancer due to high radiation dose. We developed in vitro skin models of different complexity to investigate UV-light associated skin damage. Substances and their phototoxic, sun protective or photo-sensitizing potential can be analyzed to prevent white skin cancer. Y1 - 2015 U6 - http://dx.doi.org/10.1007/s12268-015-0557-z SN - 0947-0867 VL - 21 IS - 2 SP - 172 EP - 174 PB - Springer CY - Heidelberg ER - TY - JOUR A1 - Huber, Birgit A1 - Kluger, Petra T1 - Decelerating mature adipocyte dedifferentiation by media composition JF - Tissue Engineering / Part C : Methods N2 - The establishment of adipose tissue test systems is still a major challenge in the investigation of cellular and molecular interactions responsible for the pathogenesis of inflammatory diseases involving adipose tissue. Mature adipocytes are mainly involved in these pathologies, but rarely used in vitro, due to the lack of an appropriate culture medium which inhibits dedifferentiation and maintains adipocyte functionality. In our study, we showed that Dulbecco's Modified Eagle's Medium/Ham's F-12 with 10% fetal calf serum (FCS) reported for the culture of mature adipocytes favors dedifferentiation, which was accompanied by a high glycerol release, a decreasing release of leptin, and a low expression of the adipocyte marker perilipin A, but high expression of CD73 after 21 days. Optimized media containing FCS, biotin, pantothenate, insulin, and dexamethasone decelerated the dedifferentiation process. These cells showed a lower lipolysis rate, a high level of leptin release, as well as a high expression of perilipin A. CD73-positive dedifferentiated fat cells were only found in low quantity. In this work, we showed that mature adipocytes when cultured under optimized conditions could be highly valuable for adipose tissue engineering in vitro. Y1 - 2015 U6 - http://dx.doi.org/10.1089/ten.tec.2015.0166 SN - 1937-3384 VL - 21 IS - 12 SP - 1237 EP - 1245 PB - Liebert CY - Larchmont, NY ER - TY - JOUR A1 - Wenz, Annika A1 - Janke, Katharina A1 - Hoch, Eva A1 - Tovar, Günter A1 - Borchers, Kirsten A1 - Kluger, Petra T1 - Hydroxyapatite-modified gelatin bioinks for bone bioprinting JF - Bionanomaterials : journal of functional materials, biomechanics, and tissue engineering N2 - In bioprinting approaches, the choice of bioink plays an important role since it must be processable with the selected printing method, but also cytocompatible and biofunctional. Therefore, a crosslinkable gelatin-based ink was modified with hydroxyapatite (HAp) particles, representing the composite buildup of natural bone. The inks’ viscosity was significantly increased by the addition of HAp, making the material processable with extrusion-based methods. The storage moduli of the formed hydrogels rose significantly, depicting improved mechanical properties. A cytocompatibility assay revealed suitable ranges for photoinitiator and HAp concentrations. As a proof of concept, the modified ink was printed together with cells, yielding stable three-dimensional constructs containing a homogeneously distributed mineralization and viable cells. KW - additive manufacturing KW - mineralization KW - osteoblast differentiation KW - tissue engineering Y1 - 2016 U6 - http://dx.doi.org/10.1515/bnm-2015-0018 SN - 2193-0651 VL - 17 IS - 3-4 SP - 179 EP - 184 PB - De Gruyter CY - Berlin ER - TY - JOUR A1 - Huber, Birgit A1 - Czaja, Alina Maria A1 - Kluger, Petra T1 - Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering JF - Cell biology international N2 - The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used. KW - dedifferentiation KW - EGF KW - endothelial cells KW - hydrocortisone KW - in vitro co-culture KW - mature adipocytes Y1 - 2016 U6 - http://dx.doi.org/10.1002/cbin.10595 SN - 1065-6995 VL - 40 IS - 5 SP - 569 EP - 578 PB - Wiley-Blackwell CY - Hoboken, NJ ER -