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Adipose-derived stromal cells (ASC) are a promising alternative cell source to chondrocytes as well as to bone marrow-derived mesenchymal stromal cells (BMSC) in cartilage tissue engineering and repair. Here we describe ASC isolation from liposuction by-products by collagenase-based tissue digestion combined with cell filtration and followed by monolayer attachment and expansion culture. Quality control requires confirmation of correct surface marker expression and multilineage differentiation potential by a trilineage differentiation assay.

Re-directing mesenchymal stromal cell (MSC) chondrogenesis towards a non-hypertrophic articular chondrocyte-(AC)-like phenotype is important for improving articular cartilage neogenesis to enhance clinical cartilage repair strategies. This study is the first to demonstrate that high levels of non-canonical WNT5A followed by WNT11 and LEF1 discriminated MSC chondrogenesis from AC re-differentiation. Moreover, β-catenin seemed incompletely silenced in differentiating MSCs, which altogether suggested a role for WNT signaling in hypertrophic MSC differentiation. WNT inhibition with the small molecule IWP-2 supported MSC chondrogenesis according to elevated proteoglycan deposition and reduced the characteristic upregulation of BMP4, BMP7 and their target ID1, as well as IHH and its target GLI1 observed during endochondral differentiation. Along with the pro-hypertrophic transcription factor MEF2C, multiple hypertrophic downstream targets including IBSP and alkaline phosphatase activity were reduced by IWP-2, demonstrating that WNT activity drives BMP and hedgehog upregulation, and MSC hypertrophy. WNT inhibition almost matched the strong anti-hypertrophic capacity of pulsed parathyroid hormone-related protein application, and both outperformed suppression of BMP signaling with dorsomorphin, which also reduced cartilage matrix deposition. Yet, hypertrophic marker expression under IWP-2 remained above AC level, and in vivo mineralization and ectopic bone formation were reduced but not eliminated. Overall, the strong anti-hypertrophic effects of IWP-2 involved inhibition but not silencing of pro-hypertrophic BMP and IHH pathways, and more advanced silencing of WNT activity as well as combined application of IHH or BMP antagonists should next be considered to install articular cartilage neogenesis from human MSCs.

The incidence of musculoskeletal diseases is steadily increasing with aging of the population. In the past years, extracellular vesicles (EVs) have gained attention in musculoskeletal research. EVs have been associated with various musculoskeletal pathologies as well as suggested as treatment option. EVs play a pivotal role in communication between cells and their environment. Thereby, the EV cargo is highly dependent on their cellular origin. In this review, we summarize putative mechanisms by which EVs can contribute to musculoskeletal tissue homeostasis, regeneration and disease, in particular matrix remodeling and mineralization, pro-angiogenic effects and immunomodulatory activities. Mesenchymal stromal cells (MSCs) present the most frequently used cell source for EV generation for musculoskeletal applications, and herein we discuss how the MSC phenotype can influence the cargo and thus the regenerative potential of EVs. Induced pluripotent stem cell-derived mesenchymal progenitor cells (iMPs) may overcome current limitations of MSCs, and iMP-derived EVs are discussed as an alternative strategy. In the last part of the article, we focus on therapeutic applications of EVs and discuss both practical considerations for EV production and the current state of EV-based therapies.

Tissue engineering approaches for reconstructing full-depth cartilage defects need to comprise a zone of calcified cartilage to tightly anchor cartilage constructs into the subchondral bone. Here, we investigated whether growth and differentiation factor-5-(GDF-5)-augmented fibrin hydrogel can induce a calcified cartilage-layer in vitro that seamlessly connects cartilage-relevant biomaterials with bone tissue. Human bone marrow stromal cells (BMSCs) were embedded in fibrin hydrogel and subjected to chondrogenesis with TGF-β with or without GDF-5 before constructs were implanted subcutaneously into SCID mice. A novel layered ectopic in vivo model was developed and GDF-5-augmented fibrin with BMSCs was used to glue hydrogel and collagen constructs onto bone disks to investigate formation of a calcified cartilage connecting zone. GDF-5 significantly enhanced ALP activity during in vitro chondrogenesis while ACAN and COL2A1 mRNA, proteoglycan-, collagen-type-II- and collagen-type-X-deposition remained similar to controls. Pellets pretreated with GDF-5 mineralized faster in vivo and formed more ectopic bone. In the novel layered ectopic model, GDF-5 strongly supported calcified cartilage formation that seamlessly connected with the bone. Pro-chondrogenic and pro-hypertrophic activity makes GDF-5-augmented fibrin an attractive bioactive hydrogel with high potential to stimulate a calcified cartilage connecting zone in situ that might promote integration of cartilage scaffolds with bone. Thus, GDF-5-augmented fibrin hydrogel promises to overcome poor fixation of biomaterials in cartilage defects facilitating their long-term regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2214-2224, 2018.

Current therapies involving chondrocytes or mesenchymal stromal cells (MSCs) remain inefficient in restoring cartilage properties upon injury. The induced pluripotent stem-cell (iPSC)-derived mesenchymal progenitor cells (iMPCs) have been put forward as a promising alternative cell source due to their high proliferation and differentiation potential. However, the observed cell loss during in vitro chondrogenesis is currently a bottleneck in establishing articular chondrocyte generation from iPSCs. In a search for candidate mechanisms underlying the low iPSC-derived cartilage tissue yield, global transcriptomes were compared between iMPCs and MSCs and the cell properties were analyzed via a condensation assay. The iMPCs had a more juvenile mesenchymal gene signature than MSCs with less myofibroblast-like characteristics, including significantly lower ECM- and integrin-ligand-related as well as lower α-smooth-muscle-actin expression. This correlated with less substrate and more cell-cell adhesion, impaired aggregate formation and consequently inferior cohesive tissue properties of the iMPC-pellets. Along lower expression of pro-survival ECM molecules, like decorin, collagen VI, lumican and laminin, the iMPC populations had significantly less active ERK1/2 compared to MSCs. Overall, this study proposes that this ECM and integrin-ligand shortage, together with insufficient pro-survival ERK1/2-activity, explains the loss of a non-aggregating iMPC sub-fraction during pellet formation and reduced survival of cells in early pellets. Enhancing ECM production and related signaling in iMPCs may be a promising new means to enrich the instructive microenvironment with pro-survival cues allowing to improve the final cartilage tissue yield from iPSCs.

Bioactive functional scaffolds are essential for support of cell-based strategies to improve bone regeneration. Adipose-tissue-derived-stromal cells (ASC) are more accessible multipotent cells with faster proliferation than bone-marrow-derived-stromal-cells (BMSC) having potential to replace BMSC for therapeutic stimulation of bone-defect healing. Their osteogenic potential is, however, lower compared to BMSC, a deficit that may be overcome in growth factor-rich orthotopic bone defects with enhanced bone-conductive scaffolds. Objective of this study was to compare the therapeutic potency of human ASC and BMSC for bone regeneration on a novel nanoparticulate β-TCP/collagen-carrier (β-TNC). Cytotoxicity of β-TCP nanoparticles and multilineage differentiation of cells were characterized in vitro. Cell-seeded β-TNC versus cell-free controls were implanted into 4 mm calvarial bone-defects in immunodeficient mice and bone healing was quantified by µCT at 4 and 8 weeks. Tissue-quality and cell-origin were assessed by histology. β-TNC was non-toxic, radiolucent and biocompatible, lent excellent support for human cell persistence and allowed formation of human bone tissue by BMSC but not ASC. Opposite to BMSC, ASC-grafting significantly inhibited calvarial bone healing compared to controls. Bone formation progressed significantly from 4 to 8 weeks only in BMSC and controls yielding 5.6-fold more mineralized tissue in BMSC versus ASC-treated defects. Conclusively, β-TNC was simple to generate, biocompatible, osteoconductive, and stimulated osteogenicity of BMSC to enhance calvarial defect healing while ASC had negative effects. Thus, an orthotopic environment and β-TNC could not compensate for cell-autonomous deficits of ASC which should systematically be considered when choosing the right cell source for tissue engineering-based stimulation of bone regeneration. Statement of Significance Bone-marrow-derived-stromal cells (BMSC) implanted on bone replacement materials can support bone defect healing and adipose-tissue-derived-stromal cells (ASC) being more accessible and better proliferating are considered as alternate source. This first standardized comparison of the bone regeneration potency of human ASC and BMSC was performed on a novel nanoparticular β-TCP-enriched collagen-carrier (β-TNC) designed to overcome the known inferior osteogenicity of ASC. β-TNC was non-toxic, biocompatible and osteoconductive supporting human bone formation and defect-closure by BMSC but not ASC. Long-term cell-persistence and the distinct secretome of ASC appear as main reasons why ASC inhibited bone healing opposite to BMSC. Overall, ASC-grafting is at considerable risk of producing negative effects on bone-healing while no such risks are known for BMSC.

Human mesenchymal stromal cells (hMSC) differentiating toward the chondrogenic lineage recapitulate successive phases of embryonic chondrocyte maturation developing from progenitor cells to hypertrophic chondrocytes. Osteoarthritic cartilage is characterized by an alteration in chondrocyte metabolism and upregulation of hypertrophic differentiation markers. A number of studies point toward a functional role for microRNAs (miRs) in controlling chondrocyte differentiation and development of osteoarthritis (OA). However, information on miRs that may regulate a specific phase of chondrocyte maturation, especially hypertrophy, is lacking. We here aimed to unravel miR profiles modulated during chondrogenesis of hMSC to obtain new differentiation markers and potential new targets relevant for differentiation outcome and OA development. hMSC were subjected to transforming growth factor-β (TGF-β)-driven chondrogenesis and miR profiles were determined by microarray analysis at distinct developmental time points. Expression of selected miRs was compared to cultures lacking chondrogenesis and to redifferentiated nonhypertrophic articular chondrocytes. Among 1349 probed miRs, 553 were expressed and 169 (31%) were significantly regulated during chondrogenesis. Hierarchical clustering identified specific miR expression patterns representative for MSC, prechondrocytes, chondroblasts, chondrocytes, and hypertrophic chondrocytes, respectively. Regulation of miR-181 family members allowed discrimination of successive differentiation stages. Levels of several miRs, including miR-23b, miR-140, miR-181, and miR-210 positively correlated with successful chondrocyte formation. Hypertrophic MSC-derived chondrocytes and nonhypertrophic articular chondrocytes showed differential expression of miR-181a, miR-210, and miR-31, but not miR-148a implicated in COL10A1-regulation. We conclude that the here identified stage-dependent miR clusters may have imperative functions during chondrocyte differentiation providing novel diagnostic tools and targets of potential relevance for OA development.

Tissue engineering approaches for reconstructing full-depth cartilage defects need to comprise a zone of calcified cartilage to tightly anchor cartilage constructs into the subchondral bone. Here, we investigated whether growth and differentiation factor-5-(GDF-5)-augmented fibrin hydrogel can induce a calcified cartilage-layer in vitro that seamlessly connects cartilage-relevant biomaterials with bone tissue. Human bone marrow stromal cells (BMSCs) were embedded in fibrin hydrogel and subjected to chondrogenesis with TGF-β with or without GDF-5 before constructs were implanted subcutaneously into SCID mice. A novel layered ectopic in vivo model was developed and GDF-5-augmented fibrin with BMSCs was used to glue hydrogel and collagen constructs onto bone disks to investigate formation of a calcified cartilage connecting zone. GDF-5 significantly enhanced ALP activity during in vitro chondrogenesis while ACAN and COL2A1 mRNA, proteoglycan-, collagen-type-II- and collagen-type-X-deposition remained similar to controls. Pellets pretreated with GDF-5 mineralized faster in vivo and formed more ectopic bone. In the novel layered ectopic model, GDF-5 strongly supported calcified cartilage formation that seamlessly connected with the bone. Pro-chondrogenic and pro-hypertrophic activity makes GDF-5-augmented fibrin an attractive bioactive hydrogel with high potential to stimulate a calcified cartilage connecting zone in situ that might promote integration of cartilage scaffolds with bone. Thus, GDF-5-augmented fibrin hydrogel promises to overcome poor fixation of biomaterials in cartilage defects facilitating their long-term regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2214-2224, 2018.

Mesenchymal stem cells (MSCs) have a high potential for therapeutic efficacy in treating diverse musculoskeletal injuries and cardiovascular diseases, and for ameliorating the severity of graft-versus-host and autoimmune diseases. While most of these clinical applications require substantial cell quantities, the number of MSCs that can be obtained initially from a single donor is limited. Reports on the derivation of MSC-like cells from pluripotent stem cells (PSCs) are, thus, of interest, as the infinite proliferative capacity of PSCs opens the possibility to generate large amounts of uniform batches of MSCs. However, characterization of such MSC-like cells is currently inadequate, especially with regard to the question of whether these cells are equivalent or identical to MSCs. In this study, we have derived MSC-like cells [induced PSC-derived MSC-like progenitor cells (iMPCs)] using four different methodologies from a newly established induced PSC line reprogrammed from human bone marrow stromal cells (BMSCs), and compared the iMPCs directly with the originating parental BMSCs. The iMPCs exhibited typical MSC/fibroblastic morphology and MSC-typical surface marker profile, and they were capable of differentiation in vitro along the osteogenic, chondrogenic, and adipogenic lineages. However, compared with the parental BMSCs, iMPCs displayed a unique expression pattern of mesenchymal and pluripotency genes and were less responsive to traditional BMSC differentiation protocols. We, therefore, conclude that iMPCs generated from PSCs via spontaneous differentiation represent a distinct population of cells which exhibit MSC-like characteristics.

Induced pluripotency has attracted enormous scientific and public attention and has quickly entered all fields of research not only those that had so far lacked an easily accessible cell source for cell therapies and in vitro models. The attractiveness of induced pluripotent stem (iPS) cells for cartilage regeneration roots from their immense expandability and their intrinsic ability to give rise to any adult tissue including stable hyaline cartilage. Thus, iPS cells offer to overcome the extremely limited supply of human articular chondrocytes and the restricted differentiation capacity of mesenchymal stem cells from bone marrow or adipose tissue that differentiate along the endochondral pathway and form mineralized bone upon ectopic implantation. Beyond being a potential alternative cell source for articular chondrocyte implantation, iPS cells are particularly promising for in vitro modeling of genetic diseases and for drug testing. Reprogramming patient-specific cells with a genetic predisposition and engineering disease-specific genetic variations into healthy control iPS cells promises to recapitulate “diseases in a dish” more realistically than immortalized human cell lines and will be an invaluable complementation for animal models. Whether iPS cells will satisfy these tremendous expectations will depend on our ability to upscale iPS cell culture, to derive sufficient amounts of relevant cell types like chondrocytes from iPS cells with acceptable efforts, and to find clinically safe reprogramming techniques for iPS cell-based therapies.

Cartilage degeneration in the course of osteoarthritis (OA) is associated with an alteration in chondrocyte metabolism. In order to identify molecules representing putative key regulators for diagnosis and therapeutic intervention, we analyzed gene expression and microRNA (miR) levels in OA and normal knee cartilage using a customized cartilage cDNA array and quantitative RT-PCR. Among newly identified candidate molecules, H19, IGF2, and ITM2A were significantly elevated in OA compared to normal cartilage. H19 is an imprinted maternally expressed gene influencing IGF2 expression, whose transcript is a long noncoding (lnc) RNA of unknown biological function harboring the miR-675. H19 and IGF2 mRNA levels did not correlate significantly within cartilage samples suggesting that deregulation by imprinting effects are unlikely. A significant correlation was, however, observed for H19, COL2A1, and miR-675 expression levels in OA tissue, and functional regulation of these candidate molecules was assessed under anabolic and catabolic conditions. Culture of chondrocytes under hypoxic signaling showed co-upregulation of H19, COL2A1, and miRNA-675 levels in close correlation. Proinflammatory cytokines IL-1β and TNF-α downregulated COL2A1, H19, and miR-675 significantly without close statistical correlation. In conclusion, this is the first report demonstrating deregulation of an lncRNA and its encoded miR in the context of OA-affected cartilage. Stress-induced regulation of H19 expression by hypoxic signaling and inflammation suggests that lncRNA H19 acts as a metabolic correlate in cartilage and cultured chondrocytes, while the miR-675 may indirectly influence COL2A1 levels. H19 may not only be an attractive marker for cell anabolism but also a potential target to stimulate cartilage recovery.

Mechanical strain has become an important tool in tissue engineering for progenitor cell differentiation. Furthermore, it is used to enhance the mechanical properties of engineered tissue constructs. Although strain amplitude and frequency are well investigated and optimal values are known; application of various strain schemes regarding duration and repetition are not described in literature. In this study, we therefore applied singular and repetitive cyclic strain (1 Hz, 5%) of 15 min short-time strain and longer strain durations up to 8 h. Additionally, a gradually increasing strain scheme starting with short-time strain and consecutive elongated strain periods was applied. The cultivation surface was planar silicone on one hand and a three-dimensionally structured collagen I mesh on the other hand. Adipose tissue–derived mesenchymal stem cells and an osteogenic model cell line (MG-63) were exposed to these strain regimes and post-strain cell viability, osteogenic marker gene expression, and matrix mineralization were investigated. Upregulation of alkaline phosphatase, osteocalcin, osteopontin, and BMP-2/4 revealed that even short-time strain can enhance osteogenic differentiation. Elongation and repetition of strain, however, resulted in a decline of the observed short-time strain effects, which we interpret as positively induced cellular adaptation to the mechanically active surroundings. With regard to cellular adaptation, the gradually increasing strain scheme was especially advantageous. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010

The aim of this study was the transformation of the macroporous zirconium dioxide ceramic Sponceram® into a biomimetic composite material. To enhance the adhesion of cells and to induce their differentiation into osteoblasts poly-L-lysine and BMP-2 were coupled to polymers and copolymers based on 2-deoxy-N-methacrylamido-D-glucose (ox.p(MAG) and p(MVA)) used as spacer, which were adsorbed onto the ceramic surface. The development of the composite materials was validated step by step qualitatively and quantitatively. The bioactive potential of the composite materials was tested under static and dynamic conditions using an osteoblastic model cell line and human mesenchymal stem cells. Both composite materials showed potential to enhance the adhesion of cells in the first 10 days of their cultivation. One of the composite materials, namely Sponceram®/ox.p(MAG)-BMP-2, was tested into a rotating-bed bioreactor with regard to its osteogenic differentiation-inducing potential. Compared with Sponceram® modified with BMP-2 without a polymer spacer, it showed increased expression of osteogenic markers determined by PCR analysis. In summary, the in vitro testing of the developed composite materials demonstrated a promising potential for their application as biomimetic scaffold materials with controllable properties. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009

This work is part of a general effort to demonstrate the effect of the bulk microstructure of titanium as a model bone implant material on viability of osteoblasts (bone-forming cells). The objective of this work was to study the proliferation of preosteoblastic MC3T3-E1 cells extracted from mice embryos on commercial purity titanium substrates. Two distinct states of titanium were considered: as-received material with an average grain size of 4.5 microm and that processed by equal channel angular pressing (ECAP), with an average grain size of 200 nm. We report the first results of an in vitro study into the effect of this extreme grain refinement on viability and proliferation of MC3T3-E1 cells. By means of MTT assays it was demonstrated that ECAP processing of titanium enhances MC3T3-E1 culture proliferation in a spectacular way. This finding suggests that bone implants made from ECAP processed titanium may promote bone tissue growth.

Therapeutische Ansatze der Regenerativen Medizin lassen sich, unabhangig vom Fachgebiet, der Zelltherapie, dem Tissue Engineering und der In-situ-Regeneration zuordnen. Dabei kommt der Regenerativen Orthopadie oft eine Vorreiterrolle auf dem Weg zur klinischen Anwendung zu. In der Zelltherapie sollen primare Zellen durch adulte mesenchymale Stammzellen mit nahezu unbegrenzter Regenerationskapazitat ersetzt werden; eine ausgereiftere Strukturgebung von Biomaterialien ermoglicht im Tissue Engineering die gezielte Steuerung von Zellform und Gewebeaufbau, bei der In-situ-Regeneration steht die gezielte Vermittlung von Informationen an Zellen uber intelligente Biomaterialien im Mittelpunkt der Forschung. Diese neuen Ansatze werden es moglich machen, bei der Wiederherstellung von Strukturen und Funktionen beschadigter oder dysfunktionaler Gewebe immer haufiger eine nachhaltige Verbesserung im Sinne einer echten Regeneration anstelle einer akzeptablen Reparatur zu erzielen.

For the long-term objective to engineer a functional bone construct, we applied unidirectional cyclic mechanical strain to human bone marrow stromal cells. Two strain regimes were applied comprising single and repetitive strain, respectively. For the single strain, we applied 15 and 60 min of mechanical load (1 Hz, 5% elongation). Increased proliferation and type I collagen levels were detected after 15- and 60-min load accompanied by increased type III collagen production after 60 min of strain. To study a possible amplification of effects, long time strain on 3 consecutive days, with every day 8 h strain duration, was applied in order to induce persistent and evident cellular reactions. Furthermore, an increased calcification was observed. The observed changes were not associated with changes in p38, extracellular signal regulated kinase or c-jun N-terminal kinase activation (Western Blot). In conclusion, application of a single strain period of up to 60 min is not sufficient to induce persistent cellular reactions. Fifteen minutes seems to induce beneficial effects, whereas 60 min of strain induces collagen type III (as a sign of scar formation). Repetition of strain (3 × 8 h) induced enhanced differentiation without detrimental side effects. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009

In this study, different collagen and ceramic based materials were characterized concerning their mechanical and biocompatible properties. Therefore, they were tested in static and dynamic cultivation using human mesenchymal stem cells derived from adipose tissue. Zirconia based macroporous ceramic materials with different BET surface areas (Sponceram® 30/90, Sponceram® 30/145) and surface coatings (Hydroxyapatit, Titandioxide: Sponceram®/HA, Sponceram®/Ti) were mechanically evaluated regarding their maximal tension, maximal deformation and their permeability. The maximal tension and maximal deformation of the different ceramic materials were comparable though the permeability of the uncoated ceramic with the largest BET surface area (Sponceram® 30/90) was significantly increased compared to the other ceramics. A porous collagen matrix (Matristypt®) and a porous collagen-elastin-matrix (Matriderm®) were similarly evaluated concerning their maximal tension and maximal deformation and compared with decellularized skin. The maximal deformations of the porous collagen materials were comparable but significantly increased compared to decellularized skin. The maximal tension of the porous matrices was five times lower than the maximal tension of decellularized skin. For the evaluation for cell culture applications, Sponceram®, Sponceram®/HA and two collagen meshes (Matristypt®, Matriderm®) were seeded with human mesenchymal stem cells. Sponceram® was compared to β-Tricalciumphosphate. The scaffolds were cultivated under static conditions over a time period of 14 days or 21 days respectively. The cell proliferation was examined in regular intervals using MTT assay. The cells growing on the collagen-elastin matrix showed increased proliferation in comparison to the cells grown on the pure collagen matrix. The ceramic materials tested statically in cell culture (Sponceram®, Sponceram®/HA) did not show different influence on the proliferation of seeded cells but it was increased compared to the proliferation of the cells seeded on β-Tricalciumphosophate. In a rotating bed bioreactor system (Z®RP system, Zellwerk GmbH) the human mesenchymal stem cells were cultured under dynamic conditions on Sponceram® over a time period of 47 days using osteogenic differentiation medium. Continuous increasing glucose consumption and lactate production gives evidence of good cell proliferation. After cultivation, the Sponceram® discs were stained with von kossa and alizarin red which showed matrix calcification. In summary, the tested collagen- and ceramic materials are applicable for the cell culture applications whereas the ceramic materials are presenting a promising approach for bone tissue engineering implementations. Neue Kollagen- und Keramikbasierte 3D-Biomaterialien fur das Tissue Engineering In dieser Studie wurden unterschiedliche kollagen- und keramikbasierte Matrixmaterialien in Bezug auf ihre mechanischen und biokompatiblen Eigenschaften charakterisiert. Zirkoniumdioxid basierte makroporose Keramikmaterialien mit unterschiedlichen BET-Oberflachen (Sponceram® 30/90, Sponceram® 30/145) sowie Oberflachenbeschichtungen (Hydroxylapatit, Titandioxid: Sponceram®/HA, Sponceram®/Ti) wurden mit Hilfe von Kompressions- und Perfusionsmessungen hinsichtlich ihrer maximalen Verformbarkeit und ihrer maximalen Spannungswerte sowie ihrer Permeabilitat untersucht. Die maximalen Verformbarkeiten und Spannungswerte der unterschiedlichen Keramiken variierten dabei nicht wesentlich, jedoch war die Permeabilitat der unbeschichteten Keramik mit der grosten BET-Oberflache (Sponceram® 30/90) signifikant hoher als die der anderen Keramiken. Eine Kollagen-Matrix (Matristypt®) sowie eine Kollagen-Elastin-Matrix (Matriderm®) wurden ebenso hinsichtlich ihrer maximalen Verformbarkeit sowie ihrer maximalen Spannungswerte charakterisiert und mit den Werten dezellularisierter Haut verglichen. Die maximale Verformbarkeit der Kollagen-Materialien zeigte sich dabei als vergleichbar, jedoch signifikant hoher als die der dezellularisierten Haut. Die maximal erreichten Spannungswerte unterschritten die der dezellularisierten Haut um das Funffache. Sponceram®, Sponceram®/HA und die Kollagenmaterialien wurden zur Testung fur die Anwendung in der Zellkultur zunachst mit humanen mesenchymalen Stammzellen besiedelt und unter statischen Bedingungen uber einen Zeitraum von 14 bzw. 21 Tagen kultiviert. β-Tricalciumphosphat wurde als Referenzmaterial zu Sponceram® getestet. Zur Evaluation der Zellproliferation wurde in regelmasigen Abstanden ein MTT-Test durchgefuhrt. Dabei zeigten die Zellen auf der Kollagen-Elastin-Matrix eine erhohte Viabilitat gegenuber denen auf der reinen Kollagen-Matrix. Die getesteten Keramiken (Sponceram®, Sponceram®/HA) zeigten keinen unterschiedlichen Einfluss auf die Proliferation der ausgesaten Zellen, jedoch war diese gegenuber der Proliferation von auf β-Tricalciumphosphat gewachsenen Zellen erhoht. In einem Drehbettreaktor mit Perfusionsbetrieb wurden humane mesenchymalen Stammzellen auf Sponceram® uber einen Zeitraum von 47 Tagen unter Verwendung von osteogenem Differenzierungsmedium dynamisch kultiviert. Eine kontinuierlich zunehmende Glucoseverbrauchs- und Laktatproduktionsrate wahrend der Kultivierung zeigten eine gute Zellproliferation. Nach beendeter Kultivierung wurden von Kossa- und Alizarinrotfarbungen durchgefuhrt, die eine Matrix-Kalzifizierung belegten. Zusammenfassend zeigten die untersuchten Kollagen- und Keramikmaterialien sehr gute Eignung fur die Anwendung in der Zellkultur, wobei die Keramiken einen viel versprechenden Ansatz fur das Tissue Engineering von Knochen darstellen.

Bioreaktoren sind zu einem unverzichtbaren Hilfsmittel fur das Tissue Engineering geworden, da hier Konditionen geschaffen werden konnen, unter denen sich in vitro 3D-Gewebekonstrukte unter kontrollierten und reproduzierbaren Bedingungen herstellen lassen. Der Z® RP-Bioreaktor in Verbindung mit den hochporosen Sponceram®-Tragermaterialien erlaubt eine dreidimensionale dynamische Langzeit-Kultivierung unterschiedlichster Zellarten. Dadurch wird die Herstellung von autologem Ersatzgewebe fur das Gebiet des Tissue Engineering ermoglicht.

In this study the methods of synthesis of hydrophilic aldehyde-bearing polymers and copolymers based on 2-deoxy-2-methacrylamido-D-glucose have been developed. Polymers with controllable aldehyde group content were synthesized via free-radical polymerization and subsequent polymeranalogous modification. The water-soluble polymers obtained were investigated in view of their capacity to be adsorbed on two commercial mineral supports used in bone tissue engineering. Besides, cytotoxicity of synthesized polymers was tested. 2008 Wiley Periodicals, Inc. J Appl Polym Sci 108: 2386-2397, 2008

Induced pluripotent stem cells (iPSCs) are an attractive cell source for cartilage regeneration, but current in vitro chondrogenic differentiation protocols yield insufficient results. In search for shortcomings of iPSC chondrogenesis, this study investigated whether SOX9 protein was adequately regulated during multiphase chondrogenic differentiation of two human iPSC lines in a comparable manner like during mesenchymal stromal cell (MSC) chondrogenesis. Upon generation of intermediate mesenchymal progenitor cells (iMPCs), SOX9 was induced and reached variable protein levels compared to MSCs. Along with an altered condensation behavior, iMPC cartilage formation was less robust compared to MSCs and better in the iMPC line with higher SOX9 protein levels. Despite efficient Smad-2/3 phosphorylation, TGF-v-driven chondrogenic stimulation downregulated SOX9 protein in iMPCs rather than increasing levels like in MSCs. Chondrogenesis was further improved by cotreatment with TGF-v + BMP-4, which appeared to shorten the duration of the SOX9 protein decline. However, this was insufficient to overcome heterogenic outcome and came at the expense of undesired hypertrophy. In iMPCs, but not MSCs, high levels of the SOX9-antagonizing hsa-miR-145 correlated with low SOX9 protein quantity. Thus, considerable iMPC heterogeneity with variable SOX9 protein levels, an altered condensation pattern, and low early SOX9 inducibility appeared as critical shortcomings of iPSC chondrogenesis. We suggest consistent quality of intermediate cell populations with high SOX9 protein induction as important indicators to obtain robust cartilage formation from iPSCs. The impact of this study is the identification of a SOX9 protein regulation opposite to MSC chondrogenesis that will now enable a selective adaptation of the currently limited protocols to the specific needs of iPSCs.

Xenogeneic or allogeneic chondrocytes hold great potential to build up new cartilage in vivo. However, immune rejection is a major concern for the utility of universal donor-derived cells. In order to verify the reported immune privilege of chondrocytes in vivo, the aim of this study was to assess engraftment of human articular chondrocytes (HAC) in minipig knee cartilage defects and their contribution to cartilage regeneration. HAC were transplanted matrix-assisted within two hydrogels into full-thickness cartilage defects of minipigs or implanted ectopically into immune deficient mice to assess redifferentiation capacity. At 2 and 4 weeks after surgery, cell-persistence and host cell invasion were monitored by species-specific in situ hybridization and RT-PCR. Early tissue regeneration was evaluated by histomorphometry and a modified O’Driscoll score. HAC capable of successful in vivo chondrogenic redifferentiation persisted at ectopic sites for 4 weeks in both carrier materials. Early defect regeneration involved extensive host cell invasion and a decline of HAC to less than 5 % of initial cell numbers in 6/12 defects within 2 weeks. Few clusters of persisting HAC within collagen type II-rich tissue were surrounded by porcine macrophages. Four weeks after cell transplantation, most of the defects contained well-integrated cell-rich tissue free of human cells with no apparent difference between hydrogel carriers. In summary, HAC failed to engraft in porcine articular cartilage defects despite their ability for successful in vivo redifferentiation. The co-localization of macrophages to hydrogel-implanted HAC suggests active graft rejection without evidence for an immune-privileged status of xenogeneic chondrocytes in a large animal joint.

Structural extracellular matrix molecules gain increasing attention as scaffolds for cartilage tissue engineering owing to their natural role as a growth factor repository. We recently observed that a collagen-type I/III (Col-I/III) matrix, human recombinant transforming growth factor-beta (TGF-β) protein, and fibrin hydrogel (FG) combined to a biphasic construct provided sufficient long-term TGF-β support to drive in vitro chondrogenesis of human mesenchymal stem cells (hMSC). Here we ask whether FG and Col-I/III can both retain TGF-β, describe the influence of cell seeding on TGF-β release, and compare the molecular path of hMSC chondrogenic differentiation under soluble versus local TGF-β supply. Release of growth factor from scaffolds augmented with increasing amounts of TGF-β was analyzed over 7 days and chondrogenesis was assessed over 42 days. Low TGF-β release rates from Col-I/III as opposed to higher release from FG indicated that both molecules retained TGF-β, with Col-I/III being the superior storage component. Cell seeding enhanced TGF-β retention in FG by about threefold and almost stopped release beyond 24 h. TGF-β remained bioactive and supported MSC chondrogenesis without impairing the amount of proteoglycan and collagen-type II deposition per cell and per construct compared to standard scaffold-free MSC pellets supplied with soluble TGF-β. Local TGF-β, however, mediated lower cell content, less collagen-type X relative to collagen-type II deposition and no matrix metalloproteinase-13 up-regulation. In conclusion, cells quickly halted release of local TGF-β from FG, turning FG and Col-I/III into attractive TGF-β repositories capable to drive full hMSC chondrogenesis, but via a modulated differentiation pathway. Since only part of the changes was reproduced by transient soluble TGF-β supply, release kinetics alone could not explain the molecular differences, suggesting that local TGF-β acts distinct from its soluble counterpart.

The aim of this work was the online measurement of pH and O2 values during the mechanical stimulations of cells. In different studies it was demonstrated that cells respond to mechanical strain. But the online monitoring of cells in bioreactors for mechanical stimulation remains difficult. In this study a sensor system from PreSens GmbH (Regensburg) should be integrated and tested in self developed strain apparatus. The principle of the sensors based on fluorescence measurement. The excitation of the fluorescent dye occurred through an optic fiber, whereas the fluorescent light coming back through the fiber is detected. The fluorescent dye is excited by LED and the fluorescent light is detected by PMT (photomultiplier tube).

The development of bone tissue engineering depends on the availability of suitable biomaterials, a well-defined and controlled bioreactor system, and on the use of adequate cells. The biomaterial must fulfill chemical, biological, and mechanical requirements. Besides biocompatibility, the structural and flow characteristics of the biomaterial are of utmost importance for a successful dynamic cultivation of osteoblasts, since fluid percolation within the microstructure must be assured to supply to cells nutrients and waste removal. Therefore, the biomaterial must consist of a three-dimensional structure, exhibit high porosity and present an interconnected porous network. Sponceram, a ZrO(2) based porous ceramic, is characterized in the presented work with regard to its microstructural design. Intrinsic permeability is obtained through a standard Darcy's experiment, while Young's modulus is derived from a two plates stress-strain test in the linear range. Furthermore, the material is applied for the dynamic cultivation of primary osteoblasts in a newly developed rotating bed bioreactor.

The engineering of functional bone constructs ex vivo is a rapidly growing branch in the field of tissue engineering. Bone constructs generally comprise cells and a scaffold providing a supportive framework for the cells to grow as well as mechanical stability. Furthermore, mechanical stimulation has become a substantial tool in functional tissue engineering.

Large bone defects caused by tumours, infectious diseases or trauma clearly result in the medical need for bone regeneration. For this application, the development of capable scaffold materials is essential.

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three-dimensional functional tissue-like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three-dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stem cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram. Furthermore, hydroxyapatite coated Sponceram was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP-expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram compared to hydroxyapatite coated Sponceram. With this study, we present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components.

Fracture healing is a complicated process involving many growth factors, cells, and physical forces. In cases, where natural healing is not able, efforts have to be undertaken to improve healing. For this purpose, tissue engineering may be an option. In order to stimulate cells to form a bone tissue several factors are needed: cells, scaffold, and growth factors. Stem cells derived from bone marrow or adipose tissues are the most useful in this regard. The differentiation of the cells can be accelerated using mechanical stimulation. The first part of this chapter describes the influence of longitudinal strain application. The second part uses a sophisticated approach with stem cells on a newly developed biomaterial (Sponceram) in a rotating bed bioreactor with the administration of bone morphogenetic protein-2. It is shown that such an approach is able to produce bone tissue constructs. This may lead to production of larger constructs that can be used in clinical applications.