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Purpose Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage. Methods Murine chondrogenic ATDC5 cells were differentiated into the chondrogenic lineage for seven days and subsequently differentiated towards the osteogenic direction. Gene expression analysis of pluripotency, as well as chondrogenic and osteogenic markers, cell–matrix staining, and immunofluorescent staining, were performed to assess the differentiation. In addition, the effects of Wnt3a and lipopolysaccharides (LPS) on the transdifferentiation were tested by their addition to the osteogenic differentiation medium. Results Following osteogenic differentiation, chondrogenically pe-differentiated cells displayed the expression of pluripotency and osteogenic marker genes as well as alkaline phosphatase activity and a mineralized matrix. Co-expression of Col2a1 and Col1a1 after one day of osteogenic differentiation indicated that osteogenic cells had differentiated from chondrogenic cells. Wnt3a increased and LPS decreased transdifferentiation towards the osteogenic lineage. Conclusion We successfully established a rapid, standardized in vitro assay for the transdifferentiation of chondrogenic cells into osteogenic cells, which is suitable for testing the effects of different compounds on this cellular process.

Summary Objective The human matrilin-3 T303M (in mouse T298M) mutation has been proposed to predispose for osteoarthritis, but due to the lack of an appropriate animal model this hypothesis could not be tested. This study was carried out to identify pathogenic mechanisms in a transgenic mouse line by which the mutation might contribute to disease development. Methods A mouse line carrying the T298M point mutation in the Matn3 locus was generated and features of skeletal development in ageing animals were characterized by immunohistology, micro computed tomography, transmission electron microscopy and atomic force microscopy. The effect of transgenic matrilin-3 was also studied after surgically induced osteoarthritis. Results The matrilin-3 T298M mutation influences endochondral ossification and leads to larger cartilage collagen fibril diameters. This in turn leads to an increased compressive stiffness of the articular cartilage, which, upon challenge, aggravates osteoarthritis development. Conclusions The mouse matrilin-3 T298M mutation causes a predisposition for post-traumatic osteoarthritis and the corresponding knock-in mouse line therefore represents a valid model for investigating the pathogenic mechanisms involved in osteoarthritis development.

ZusammenfassungDupilumab ist ein monoklonaler Antikörper, der an die gemeinsame α‑Kette des IL(Interleukin)-4- und IL-13-Rezeptors bindet und den Th(T-Helferzelle)2-Signalweg blockiert, der bei der Entstehung des atopischen Ekzems eine Schlüsselrolle spielt. Wir berichten über den Fall eines 40-jährigen Patienten, der nach 6 Wochen Dupilumab-Therapie eine histologisch gesicherte Psoriasis entwickelte. Das eigenmächtige, abrupte Absetzen der ungewöhnlichen, nicht leitliniengerechten oralen Steroidtherapie und die Blockade des Th2-Signalwegs durch Dupilumab dürften die entscheidenden Auslösefaktoren für die erstmalige Ausbildung der Psoriasis bei unserem Patienten gewesen sein.

Energy metabolism and extracellular matrix function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single cell RNA-seq (scRNA-seq) analysis to uncover the importance of the mitochondrial respiratory chain for extracellular matrix homeostasis in mature cartilage. This tissue produces large amounts of a specialized extracellular matrix to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocytes. These changes were associated with alterations in extracellular matrix composition, a shift in collagen/non-collagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.

Previous anatomical studies have shown different functional zones in human nasal septal cartilage (NC). These zones differ in respect to histological architecture and biochemical composition. The aim of this study was to investigate the influence of these zones on the fate of stem cells from a regenerative perspective. Therefore, decellularized porcine septal cartilage was prepared and subjected to histological assessment to demonstrate its equivalence to human cartilage. Decellularized porcine NC (DPNC) exposed distinct surfaces depending on two different histological zones: the outer surface (OS), which is equivalent to the superficial zone, and the inner surface (IS), which is equivalent to the central zone. Human adipose tissue-derived stem cells (ASCs) were isolated from the abdominal fat tissue of five female patients and were seeded on the IS and OS of DPNC, respectively. Cell seeding efficiency (CSE), vitality, proliferation, migration, the production of sulfated glycosaminoglycans (sGAG) and chondrogenic differentiation capacity were evaluated by histological staining (DAPI, Phalloidin, Live-Dead), biochemical assays (alamarBlue®, PicoGreen®, DMMB) and the quantification of gene expression (qPCR). Results show that cell vitality and CSE were not influenced by DPNC zones. ASCs, however, showed a significantly higher proliferation and elevated expression of early chondrogenic differentiation, as well as fibrocartilage markers, on the OS. On the contrary, there was a significantly higher upregulation of hypertrophy marker MMP13 (p &lt, 0.0001) and GAG production (p = 0.0105) on the IS, whereas cell invasion into the three-dimensional DPNC was higher in comparison to the OS. We conclude that the zonal-dependent distinct architecture and composition of NC modulates activities of ASCs seeded on DPNC. These findings might be used for engineering of cartilage substitutes needed in facial reconstructive surgery that yield an equivalent histological and functional structure, such as native NC.

Adherence describes how a patient follows a medical regime recommended by a healthcare provider. Poor treatment adherence represents a complex and challenging problem of international healthcare systems, as it has a substantial impact on clinical outcomes and patient safety and constitutes an important financial burden. Since it is one of the most common causes of treatment failure, it is extremely important for physicians to reliably distinguish between non-adherence and non-response. This systematic review aims to summarize the current literature on treatment adherence in dermatology, focusing on chronic inflammatory skin diseases such as psoriasis, atopic dermatitis and acne. A systematic literature search was performed using the PubMed Database, including articles from 2008 to 2018. Low treatment adherence is a multidimensional phenomenon defined by the interplay of numerous factors and should under no circumstances be considered as the patient's fault alone. Factors influencing treatment adherence in dermatology include patient characteristics and beliefs, treatment efficacy and duration, administration routes, disease chronicity and the disease itself. Moreover, the quality of the physician-patient relationship including physician-time available for the patient plays an important role. Understanding patients' adherence patterns and the main drivers of non-adherence creates opportunities to improve adherence in the future. Strategies to increase treatment adherence range from reminder programs to simplifying prescriptions or educational interventions. Absolute adherence to treatment may not be realistically achievable, but efforts need to be made to raise awareness in order to maximize adherence as far as possible.

Der monoklonale Antikorper Dupilumab (Dupixent® ) ist seit September 2017 in der EU als neue systemische Therapie bei mittelschwerem bis schwerem atopischem Ekzem zugelassen. Eine Unterdruckung der IL-4- und IL-13-Signalwege durch die Anwendung von Dupilumab fuhrt zu einer Verbesserung objektiver und subjektiver Komponenten der Erkrankung. Die Blockade der IL-4Rα-Untereinheit fuhrt zu einer Reduktion der Th2-vermittelten Entzundung und zu einer Verbesserung der Hautbarriere. Die Nebenwirkungsrate unter Dupilumab ist gering. Allerdings entwickelten bis zu 28 % der Patienten unter Behandlung mit Dupilumab (gegenuber bis zu 11 % unter Placebo) eine bilaterale, milde bis moderate Konjunktivitis mit Rotung, Brennen und Fremdkorpergefuhl der Augen. Mit topischen Steroiden und topischen Calcineurininhibitoren stehen verschiedene Therapieoptionen der Dupilumab-assoziierten Konjunktivitis zur Verfugung. Dieser Artikel berichtet uber die klinische Prasentation sowie die medikamentosen und nichtmedikamentosen Therapieoptionen der klinisch hochrelevanten Dupilumab-assoziierten Konjunktivitis.

Zusammenfassung Anamnese und klinischer Befund Eine 25-jährige Patientin wurde aufgrund eines schweren atopischen Ekzems (AE) mit dem anti IL-4/IL-13-Rezeptor-Antikörper Dupilumab behandelt. Zusätzlich litt die Patientin seit 5 Jahren an einer Alopecia areata (AA). Untersuchungen und Diagnose Bei Erstvorstellung zeigte die körperliche Untersuchung ein schweres AE (Scoring Atopic Dermatitis [SCORAD] 76,0; Eczema Area and Severity Index [EASI] 37,4) mit einem Gesamt-IgE von 1108 kU/l sowie eine Alopecia areata. Therapie und Verlauf Vor einem Jahr wurde eine Behandlung mit dem anti IL-4/IL-13-Rezeptor-Antikörper Dupilumab eingeleitet. Nach 5 Monaten zeigte sich eine deutliche Besserung des AE (SCORAD 32,2; EASI 3,8), sowie ein zunehmender Haarwuchs. Nach 11 Monaten Therapiedauer blieb das AE stabil gebessert (SCORAD 27,0; EASI 2,7), zudem waren nahezu alle Haare nachgewachsen. Folgerung Die Komorbidität zwischen AE und AA ist seit Jahren bekannt, die Beeinflussung der AA durch Dupilumab ist eine neue Erkenntnis. Der vorliegende Fallbericht unterstützt die Hypothese, dass eine Hemmung der IL-4- und IL-13-Signalwege tatsächlich und längerfristig Einfluss auf den Haarwuchs bei AA hat. Bei Patienten mit AE und begleitender AA ist ein positiver Effekt auch auf die Komorbidität AA zu erwarten.

During biomineralization, the cells generating the biominerals must be able to sense the external physical stimuli exerted by the growing mineralized tissue and change their intracellular protein composition according to these stimuli. In molluscan shell, the myosin-chitin synthases have been suggested to be the link for this communication between cells and the biomaterial. Hyaluronan synthases (HAS) belong to the same enzyme family as chitin synthases. Their product hyaluronan (HA) occurs in the bone and is supposed to have a regulatory function during bone regeneration. We hypothesize that HASes’ expression and activity are controlled by fluid-induced mechanotransduction as it is known for molluscan chitin synthases. In this study, bone marrow-derived human mesenchymal stem cells (hMSCs) were exposed to fluid shear stress of 10 Pa. The RNA transcriptome was analyzed by RNA sequencing (RNAseq). HA concentrations in the supernatants were measured by ELISA. The cellular structure of hMSCs and HAS2-overexpressing hMSCs was investigated after treatment with shear stress using confocal microscopy. Fluid shear stress upregulated the expression of genes that encode proteins belonging to the HA biosynthesis and bone mineralization pathways. The HAS activity appeared to be induced. Knowledge about the regulation mechanism governing HAS expression, trafficking, enzymatic activation and quality of the HA product in hMSCs is essential to understand the biological role of HA in the bone microenvironment.

Background Cartilage shortage is a major problem in facial reconstructive surgery. Prior studies have shown that decellularized porcine nasal septal cartilage (DPNC) seeded with primary human nasal chondrocytes enabled cartilage regeneration and showed potential as a replacement material for nasal cartilage. Since adipose tissue-derived stem cells (ASCs) are easily accessible and almost abundantly available, they appear to be a promising alternative to limited chondrocytes making the combination of DPNC and ASCs a feasible approach towards clinical translation. Thus, this study was intended to investigate the interactions between ASCs and DPNC in an in vitro model. Methods DPNCs were seeded and 3D-cultured with primary human ASCs that were priorly characterized with trilineage differentiation and flow cytometry. Cell vitality and proliferation were evaluated by Live-Dead, alamarBlue, and PicoGreen assays. Chondrogenic differentiation was examined by DMMB assay and cryosectioning-based histology. Cell invasion within DPNC was visualized and quantified by fluorescent histology (DAPI, Phalloidin). Results ASCs showed good adherence to DPNC and Live-Dead assay proved their viability over 2 weeks. AlamarBlueassay showed an increase in metabolic activity compared to 2D cultures, and PicoGreen assay demonstrated an increase of cell number within DPNC over time. Biochemical assays and histology added evidence of chondrogenic differentiation of 3D-cultured ASCs under the influence of chondrogenic induction medium. Fluorescent image analysis showed a significant increase of cell-occupied areas of scaffolds over time (P Conclusions DPNC scaffolds provided a suitable environment for ASCs that allowed good cell vitality, high proliferation, and chondrogenic differentiation. Thus, the use of ASCs and DPNC yields a promising alternative to the use of primary human chondrocytes. For facial cartilage tissue engineering, we regard ASCs as an attractive alternative to human nasal chondrocytes due to their better accessibility and availability. Further research will be necessary to determine long-term effects and in vivo outcomes of ASCs and DPNC in cartilage regeneration of the face.

Adipose-derived multipotent stem/progenitor cells (ASPCs) were shown to be ideal candidates for cell-based regenerative therapies. Yet, despite their huge potential, successful clinical applications are still rare. It was suggested that the efficacy of ASPCs at the recipient site depends on the vehicle of cell delivery. In this study, for preparation of a murine critical-size nerve defect model, we assessed the commercially available fibrin gel (ARTISS) as a potential cell carrier. In a thorough in vitro analysis, we investigated cell-fibrin interactions and analyzed the distribution and the long-term behavior of ASPCs cultivated in fibrin gel under normoxic and hypoxic conditions. ASPCs attached to the surface of a thin fibrin layer (two-dimensional condition) and spread with the abundant formation of actin stress fibers. Cells cultured within a fibrin matrix (three-dimensional condition) displayed a uniform distribution and formed interconnected networks while exhibiting strong cell-matrix interactions. Using time-lapse analysis, cells were found to migrate out of the gel and subsequently proliferated robustly both under hypoxic and normoxic conditions. During 14 days of culture in fibrin gel, ASPCs showed high viability, metabolic, and remodeling activities. At the end of the culture period, the fibrin matrix was degraded entirely accompanied by an upregulation of matrix metalloproteinases. In conclusion, fibrin gel stands out as a valuable biomaterial for delivering vital and active cells to damaged tissues. As a direct proof, ASPCs carried in a fibrin matrix will be evaluated in a murine critically sized peripheral nerve repair model.

Hyaluronan (HA), a natural component of the extracellular matrix, is supposed to have a regulatory function in the stem cell niche. Bone marrow-derived human mesenchymal stem cells (hMSCs) are known to express all three hyaluronan synthases (HASes), which are responsible for HA production. HA is extruded into the extracellular matrix, but also stays bound to the plasma membrane forming a pericellular coat, which plays a key role during early cell adhesion. Since HAS isoenzymes, HAS1, HAS2 and HAS3, produce HA with different molecular weights, a difference in their role for cell adhesion is expected. Here, we transduced the immortalized hMSC cell line SCP1 to constitutively express eGFP-tagged HASes (SCP1-HAS-eGFP) by lentiviral gene transfer. The overexpression of the HAS-eGFP was shown on RNA and protein levels, HA was determined by ELISA and the stained HA-coat was analyzed using confocal microscopy. Time-lapse microscopy, spreading assay and single cell force spectroscopy using atomic force microscopy were applied to characterize adhesion of the different HAS transduced SCP1 cells. We showed in this study that HAS3 overexpressing cells formed the thickest pericellular coat compared with control or HAS1 and HAS2 transduced cells. Furthermore, SCP1-HAS3-eGFP displayed faster and stronger adhesion compared to cells overexpressing the other synthases or control cells. We conclude that overexpression of HASes in hMSCs differentially modulates their initial adhesive interactions with the substrate. This observation might be helpful in regenerative medicine goals.

Integrity of the musculoskeletal system is essential for the transfer of muscular contraction force to the associated bones. Tendons and skeletal muscles intertwine, but on a cellular level, the myotendinous junctions (MTJs) display a sharp transition zone with a highly specific molecular adaption. The function of MTJs could go beyond a mere structural role and might include homeostasis of this musculoskeletal tissue compound, thus also being involved in skeletal muscle regeneration. Repair processes recapitulate several developmental mechanisms, and as myotendinous interaction does occur already during development, MTJs could likewise contribute to muscle regeneration. Recent studies identified tendon-related, scleraxis-expressing cells that reside in close proximity to the MTJs and the muscle belly. As the muscle-specific function of these scleraxis positive cells is unknown, we compared the influence of two immortalized mesenchymal stem cell (MSC) lines&mdash, differing only by the overexpression of scleraxis&mdash, on myoblasts morphology, metabolism, migration, fusion, and alignment. Our results revealed a significant increase in myoblast fusion and metabolic activity when exposed to the secretome derived from scleraxis-overexpressing MSCs. However, we found no significant changes in myoblast migration and myofiber alignment. Further analysis of differentially expressed genes between native MSCs and scleraxis-overexpressing MSCs by RNA sequencing unraveled potential candidate genes, i.e., extracellular matrix (ECM) proteins, transmembrane receptors, or proteases that might enhance myoblast fusion. Our results suggest that musculotendinous interaction is essential for the development and healing of skeletal muscles.

Atopic eczema is a chronic recurrent inflammatory skin disease characterized by intensive pruritus and a high burden of disease. Based on a genetically determined skin barrier dysfunction, xerosis cutis and a tendency towards microbial skin infections are the leading clinical features. Mild and moderate disease manifestations are common, and usually treated with topical agents only. Treatment concepts are usually based on a combination of (i) topical basic therapy consisting of skin cleansing and barrier stabilizing emollients and (ii) topical anti-inflammatory therapy of visible skin lesions with topical corticosteroids and topical calcineurin inhibitors. Proactive therapy of the commonly affected and usually relapsing areas of skin is an important therapeutic option for long-term maintenance treatment of moderate to severe disease. Patients should be actively involved in planning of treatment, which should be adapted to individual patient factors such as age, involved body areas, type of skin lesions, as well as seasonal and climatic factors. New promising treatment options including topical phosphodiesterase inhibitors and topical Janus kinase inhibitors are currently being evaluated in clinical trials and may become a future treatment option for atopic eczema. This review article summarizes the current topical treatment options and new perspectives in the topical therapy of atopic eczema.

A novel microindentation sensor system with the potential to be integrated into arthroscopic instrumentation is presented to assess biomechanical properties of articular cartilage. The piezoelectric driven indentation device was realized by using the tip of an optical fiber with an integrated fiber Bragg grating for force sensing. The performance of the system was demonstrated by ex vivo stepwise indentation experiments performing 15 steps each 20 μm in depth on explants of bovine femoral condyle cartilage. Parameters describing the relaxation were extracted using a viscoelastic model, which includes two relaxation time constants, one in the order of 0.2–5 s and the other in the order of 10–40 s. All model parameters showed increasing values with indentation depth. The plots of the model parameters against the indentation depth suggested that the faster relaxation process was mainly influenced by the superficial zone of the articular cartilage while the middle zone cartilage contributed predominantly to the slower relaxation process. The small indenter size allowed the assessment of the spatial distribution of the mechanical characteristics of the cartilage, which was determined within a frame of 3 × 5 mm2 on each sample. A stronger dependence of the model parameters along the medial-lateral axis than the posterior-anterior direction of the knee was found. In conclusion the device presented showed the potential to discriminate between the superficial and middle zone of the articular cartilage and to assess with high resolution changes in the constitution of the tissue.

The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti‐angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament‐specific marker and anti‐angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age‐dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd − / −) mice exhibited more rapid progression of age‐related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD‐ and tendon/ligament‐related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic‐like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd − / − Chm1 − / −) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF‐derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age‐related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age‐related IVD degeneration., Cartoon highlighting the hallmarks of Tnmd−/− intervertebral disc (IVD) phenotype. Tnmd−/− mice exhibited more rapid progression of age‐related IVD degeneration. These signs include lower disc height index, smaller collagen fibrils with lower compressive stiffness in the outer annulus fibrosus, reduced multiple IVD‐ and tendon/ligament‐related gene expression, induced angiogenesis and macrophage infiltration in outer annulus fibrous, as well as more hypertrophic‐like chondrocytes in the nucleus pulposus.

The application of liposuctioned white adipose tissue (L-WAT) and adipose-derived stem cells (ADSCs) as a novel immunomodulatory treatment option is the currently subject of various clinical trials. Because it is crucial to understand the underlying therapeutic mechanisms, the latest studies focused on the immunomodulatory functions of L-WAT or ADSCs. However, studies that examine the specific transcriptional adaptation of these treatment options to an extrinsic inflammatory stimulus in an unbiased manner are scarce. The aim of this study was to compare the gene expression profile of L-WAT and ADSCs, when subjected to tumor necrosis factor alpha (TNF&alpha, ), and to identify key factors that might be therapeutically relevant when using L-WAT or ADSCs as an immuno-modulator. Fat tissue was harvested by liposuction from five human donors. ADSCs were isolated from the same donors and shortly subjected to expansion culture. L-WAT and ADSCs were treated with human recombinant TNF&alpha, to trigger a strong inflammatory response. Subsequently, an mRNA deep nextgeneration sequencing was performed to evaluate the different inflammatory responses of L-WAT and ADSCs. We found significant gene expression changes in both experimental groups after TNF&alpha, incubation. However, ADSCs showed a more homogenous gene expression profile by predominantly expressing genes involved in immunomodulatory processes such as CCL19, CCL5, TNFSF15 and IL1b when compared to L-WAT, which reacted rather heterogeneously. As RNA sequencing between L-WAT and ADSCS treated with TNF&alpha, revealed that L-WAT responded very heterogeneously to TNF&alpha, treatment, we therefore conclude that ADSCs are more reliable and predictable when used therapeutically. Our study furthermore yields insight into potential biological processes regarding immune system response, inflammatory response, and cell activation. Our results can help to better understand the different immunomodulatory effects of L-WAT and ADSCs.

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/PCP (planar cell polarity) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which β1 integrin gene is ablated at E8.0 in the notochord only or in the notochord and tail bud. These β1 integrin conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wild-type embryos with anti-β1 integrin blocking antibodies to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of β1 integrin for the establishment of PCP and convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations.

Fragestellung: Der Verlust des Nasenseptumknorpels stellt die moderne Rekonstruktionschirurgie vor große Herausforderungen. Der therapeutische Goldstandard, die autologe Knorpeltransplantation, kann zu ausgeprägten mechanischen und kosmetischen Komplikationen führen. Daher ist besonders[zum vollständigen Text gelangen Sie über die oben angegebene URL], 50. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 24. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)

Fragestellung: Eine Möglichkeit der Knorpelregeneration zur Rekonstruktion von Knorpeldefekten besteht in der Verwendung von dezellularisierter extrazellulärer Matrix. Vorversuche hatten am Menschen und im Schwein allerdings gezeigt, dass die natürliche extrazelluläre Matrix des [zum vollständigen Text gelangen Sie über die oben angegebene URL], 50. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 24. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)

The growth plate (GP) is a dynamic tissue driving bone elongation through chondrocyte proliferation, hypertrophy and matrix production. The extracellular matrix (ECM) is the major determinant of GP biomechanical properties and assumed to play a pivotal role for chondrocyte geometry and arrangement, thereby guiding proper growth plate morphogenesis and bone elongation. To elucidate the relationship between morphology and biomechanics during cartilage morphogenesis, we have investigated age-dependent structural and elastic properties of the proliferative zone of the murine GP by atomic force microscopy (AFM) from the embryonic stage to adulthood. We observed a progressive cell flattening and arrangement into columns from embryonic day 13.5 until postnatal week 2, correlating with an increasing collagen density and ECM stiffness, followed by a nearly constant cell shape, collagen density and ECM stiffness from week 2 to 4 months. At all ages, we found marked differences in the density and organization of the collagen network between the intracolumnar matrix, and the intercolumnar matrix, associated with a roughly two-fold higher stiffness of the intracolumnar matrix compared to the intercolumnar matrix. This difference in local ECM stiffness may force the cells to arrange in a columnar structure upon cell division and drive bone elongation during embryonic and juvenile development.

We have determined the sensitivity and detection limit of a new fiber Bragg grating (FBG)-based optoelectronic micro-indenter for biomechanical testing of cartilage and compared the results to indentation-type atomic force microscopy (IT-AFM) and histological staining. As test samples, we used bovine articular cartilage, which was enzymatically degraded ex vivo for five minutes using different concentrations of collagenase (5, 50, 100 and 500 &micro, g/mL) to mimic moderate extracellular matrix deterioration seen in early-stage osteoarthritis (OA). Picrosirius Red staining and polarization microscopy demonstrated gradual, concentration-dependent disorganization of the collagen fibrillar network in the superficial zone of the explants. Osteoarthritis Research Society International (OARSI) grading of histopathological changes did not discriminate between undigested and enzymatically degraded explants. IT-AFM was the most sensitive method for detecting minute changes in cartilage biomechanics induced by the lowest collagenase concentration, however, it did not distinguish different levels of cartilage degeneration for collagenase concentrations higher than 5 &micro, g/mL. The FBG micro-indenter provided a better and more precise assessment of the level of cartilage degeneration than the OARSI histological grading system but it was less sensitive at detecting mechanical changes than IT-AFM. The FBG-sensor allowed us to observe differences in cartilage biomechanics for collagenase concentrations of 100 and 500 &micro, g/mL. Our results confirm that the FBG sensor is capable of detecting small changes in articular cartilage stiffness, which may be associated with initial cartilage degeneration caused by early OA.

Summary Background Adipose-derived stem/progenitor cells (ADSPCs) are under investigation in many clinical applications for their regenerative potential in a variety of autoimmune, degenerative, and inflammatory diseases. Adipose tissue, which is mainly harvested by manual liposuction, is the main source of these ADSPCs. Objective In the past years, a variety of different liposuction devices have been commercialized. To ensure a high quality of obtained ADSPCs, it is crucial to show the advantages and disadvantages of frequently used liposuction methods. For this reason, the objective of this study was to compare ADSPCs harvested by either the suction-assisted LipiVage200-5 or the water-assisted Body-Jet system. Methods The proliferation potential of ADSPCs, harvested from 20 patients, was assessed by cumulative population doublings (cumPD), population doubling time (PDT), colony-forming units (CFU), and cell metabolism assays. To prove the multipotency of the primary isolated cells, ADSPCs were induced to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Results Our data show a significantly higher cumPD, as well as a significantly lower PDT for cells obtained by the Body-Jet system. No significant differences were found regarding the CFU efficiency and the cell metabolism. Furthermore, we showed that the adipogenic, osteogenic, and chondrogenic potential of ADSPCs is similar in both groups. Discussion/conclusion In our study, we provide evidence that the cell characteristics and the functional properties of ADSPCs isolated after liposuction with different techniques are largely similar. However, we observed a significantly higher cumPD and a slightly higher adipogenic potential in cells isolated after liposuction with the Body-Jet system. Different cannula sizes and sheer stresses in the used methods might play a role here.

The gene encoding the proteoglycan aggrecan (Agc1) is abundantly expressed in cartilage during development and adulthood, and the loss or diminished deposition of the protein results in a wide range of skeletal malformations. Furthermore, aggrecan degradation is a hallmark of cartilage degeneration occurring in osteoarthritis. In the present study, we investigated the consequences of a partial loss of aggrecan in the postnatal skeleton and in the articular cartilage of adult mice. We took advantage of the previously described Agc1tm(IRES-CreERT2) mouse line, which allows for conditional and timely-regulated deletion of floxed, cartilage-expressed genes. As previously reported, the introduction of the CreERT2 cassette in the 3&rsquo, UTR causes a disruption of the normal expression of Agc1 resulting in a hypomorphic deposition of the protein. In homozygous mice, we observed a dwarf phenotype, which persisted throughout adulthood supporting the evidence that reduced aggrecan amount impairs skeletal growth. Homozygous mice exhibited reduced proteoglycan staining of the articular cartilage at 6 and 12 months of age, increased stiffening of the extracellular matrix at six months, and developed severe cartilage erosion by 12 months. The osteoarthritis in the hypomorph mice was not accompanied by increased expression of catabolic enzymes and matrix degradation neoepitopes. These findings suggest that the degeneration found in homozygous mice is likely due to the compromised mechanical properties of the cartilage tissue upon aggrecan reduction.

In this study, the capability of a fiber optic microindenter sensor to discriminate between healthy and slightly degenerated human articular cartilage samples is demonstrated. The purely optical indenter sensor is characterized by extremely reduced dimensions (0.125 mm in diameter and 27 mm in length) in comparison to existing indenter probes offering advantages for endoscopic deployment. The indenter sensor is intended to assist the surgeon in the identification of damaged articular cartilage. From each of seven specimens of human tibia plateau three samples showing different Outerbridge grading were extracted. On each sample stress-relaxation measurements were performed with eight indentation steps, each step being 40 μm and the relaxation of the material was observed for 240 s after each step. A viscoelastic model was used to fit the relaxation and to extract the characteristic parameters according to the model. A highly significant difference in stiffness (p value

Fragestellung: Rhizarthrose verursacht Schmerzen, Schwäche und führt langfristig zu einem Beweglichkeitsverlust. Derzeitige Behandlungen lindern die Symptome nur vorübergehend und meist folgt eine Trapezektomie. Um diese Behandlungsmöglichkeiten zu erweitern, wurde von uns als regenerativer[zum vollständigen Text gelangen Sie über die oben angegebene URL], 59. Kongress der Deutschen Gesellschaft für Handchirurgie

Die Regeneration von Knorpeldefekten mit neuartiger dezellularisierter Knorpelmatrix (DECM) vom Schwein konnte bereits mit Chondrozyten erfolgreich in vitro und in vivo gezeigt werden. Der Einsatz von leicht zugänglichen Fettgewebsstammzellen (ASCs) ist allerdings deutlich schwieriger, da diese[zum vollständigen Text gelangen Sie über die oben angegebene URL], 49. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 23. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)

Long-bone growth by endochondral ossification is cooperatively accomplished by chondrocyte proliferation, hypertrophic differentiation, and appropriate secretion of collagens, glycoproteins, and proteoglycans into the extracellular matrix (ECM). Before folding and entering the secretory pathway, ECM macromolecules in general are subject to extensive posttranslational modification, orchestrated by chaperone complexes in the endoplasmic reticulum (ER). ERp57 is a member of the protein disulfide isomerase (PDI) family and facilitates correct folding of newly synthesized glycoproteins by rearrangement of native disulfide bonds. Here, we show that ERp57-dependent PDI activity is essential for postnatal skeletal growth, especially during the pubertal growth spurt characterized by intensive matrix deposition. Loss of ERp57 in growth plates of cartilage-specific ERp57 knockout mice (ERp57 KO) results in ER stress, unfolded protein response (UPR), reduced proliferation, and accelerated apoptotic cell death of chondrocytes. Together this results in a delay of long-bone growth with the following characteristics: (1) enlarged growth plates; (2) expanded hypertrophic zones; (3) retarded osteoclast recruitment; (4) delayed remodeling of the proteoglycan-rich matrix; and (5) reduced numbers of bone trabeculae. All the growth plate and bone abnormalities, however, become attenuated after the pubertal growth spurt, when protein synthesis is decelerated and, hence, ERp57 function is less essential.

Objectives: Osteoarthritis is one of the prevalent causes of disability worldwide. Due to its avascularity, and the reduced mitotic activity of adult chondrocytes together with their decreased responsiveness to anabolic growth factors, articular cartilage has limited capacity for self-repair. In this[for full text, please go to the a.m. URL], Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2017)

Fragestellung: Es ist wahrscheinlich, dass ADSPCs durch ihre antiinflammatorischen und immunsupressiven Eigenschaften den therapeutisch bedeutendsten Zellanteil im Rahmen autologer Fettinjektionen zur Behandlung der Rhizarthrose ausmachen. Daher besteht ein großes Interesse daran zu wissen, bei[zum vollständigen Text gelangen Sie über die oben angegebene URL], 58. Kongress der Deutschen Gesellschaft für Handchirurgie

Einleitung: Mesenchymale Vorläuferzellen stellen eine vielversprechende Quelle für zellbasierte Therapie im Bereich der regenerativen Medizin dar. Es ist wahrscheinlich, dass die aus Fettgewebe gewonnen adipose derived stem progenitor cells (ADSPCs) durch ihre antiinflammatorischen und immunsupressiven[zum vollständigen Text gelangen Sie über die oben angegebene URL], 48. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 55. Jahrestagung der Österreichischen Gesellschaft für Plastische, Ästhetische und Rekonstruktive Chirurgie, 22. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)

Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4&minus, /&minus, mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4&minus, mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4&minus, mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4&minus, mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column.

Integrins are cell surface receptors that connect extracellular matrix (ECM) components to the actin cytoskeleton and transmit chemical and mechanical signals into the cells through adhesion complexes. Integrin-activated downstream pathways have been implicated in the regulation of various cellular functions, including proliferation, survival, migration, and differentiation. Integrin-based attachment to the matrix plays a central role in development, tissue morphogenesis, adult tissue homeostasis, remodeling and repair, and disturbance of the ECM-integrin-cytoskeleton signaling axis often results in diseases and tissue dysfunction. Increasing amount of in vitro and in vivo evidences suggest that integrins are pivotal for proper development, function, and regeneration of skeletal tissues. In this paper, we will summarize and discuss the role of integrins in skeletogenesis and their influence on the physiology and pathophysiology of cartilage, bone, and tendon. Birth Defects Research (Part C) 102:13–36, 2014. © 2014 Wiley Periodicals, Inc.

Osteoarthritis (OA) is a leading cause of disability and source of societal cost in older adults [...]

The development and homeostasis of cartilaginous tissues are regulated by diverse microenvironmental cues including integrin-mediated interactions between chondrocytes and the extracellular matrix (ECM). Integrins are membrane receptors responsible for bi-directional communication between the cells and the surrounding by transmitting physicochemical signals through adhesion complexes. In addition, integrins are involved in sensing mechanical stress signals generated by the ECM and transduce them into the cell interior converting physical stimuli to biochemical signaling. Integrin-activated signaling cascades modulate various chondrocyte functions and play important roles in cartilage morphogenesis, homeostasis, and repair. This chapter will summarize and discuss the role of integrins in the physiology and pathophysiology of the growth plate and articular cartilage.

Fragestellung: Die Arthrose des Kniegelenkes (OA) stellt aufgrund unserer zunehmend alternden Gesellschaft eine wachsende sozioökonomische Belastung dar. Wachstumsfaktor-basierte regenerative Therapieansätze könnten eine zukünftige Behandlungsoption darstellen dem Knorpelverlust,[zum vollständigen Text gelangen Sie über die oben angegebene URL], Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2016)

Fragestellung: Unzureichende Regeneration peripherer Nerven stellt im klinischen Alltag nach wie vor ein ernsthaftes Problem dar. Daher ist es notwendig neue Therapieansätze zu entwickeln. Als Goldstandard zur Überbrückung größerer Kontinuitätslücken gilt in der klinischen[zum vollständigen Text gelangen Sie über die oben angegebene URL], 47. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 21. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)

Einleitung: Das Potenzial von multipotenten Stamm-/Vorläuferzellen aus Fettgewebe (ASPCs) für die regenerative Medizin ist gut belegt. Problematisch für den klinischen Einsatz von ASPCs ist jedoch die Zellisolation, welche oft Expansionsschritte außerhalb des OPs beinhaltet.[zum vollständigen Text gelangen Sie über die oben angegebene URL], 47. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 21. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)

The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in ex vivo systems than in in vitro cell cultures. I have therefore established an ex vivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p = 0.0083) and P2 (p

Zielsetzung: Metaphysäre Frakturen stellen insbesondere in osteoporotischem Knochen eine chirurgische Herausforderung dar. Um eine zügige Mobilisation der überwiegend geriatrischen Patienten zu gewährleisten, sind an eine Osteosynthese hohe Anforderungen zu stellen. Für eine[zum vollständigen Text gelangen Sie über die oben angegebene URL], Alterstraumatologie Kongress 2016

ObjectiveInterterritorial regions of articular cartilage matrix are rich in decorin, a small leucine-rich proteoglycan and important structural protein, also involved in many signalling events. Decorin sequesters transforming growth factor β (TGFβ), thereby regulating its activity. Here, we analysed whether increased bioavailability of TGFβ in decorin-deficient (Dcn−/−) cartilage leads to changes in biomechanical properties and resistance to osteoarthritis (OA).MethodsUnchallenged knee cartilage was analysed by atomic force microscopy (AFM) and immunohistochemistry. Active transforming growth factor β-1 (TGFβ1) content within cultured chondrocyte supernatants was measured by ELISA. Quantitative real-time (RT)-PCR was used to analyse mRNA expression of glycosaminoglycan (GAG)-modifying enzymes in C28/I2 cells following TGFβ1 treatment. In addition, OA was induced inDcn−/−and wild-type (WT) mice via forced exercise on a treadmill.ResultsAFM analysis revealed a strikingly higher compressive stiffness inDcn−/−than in WT cartilage. This was accompanied by increased negative charge and enhanced sulfation of GAG chains, but not by alterations in the levels of collagens or proteoglycan core proteins. In addition, decorin-deficient chondrocytes were shown to release more active TGFβ1. Increased TGFβ signalling led to enhancedChst11sulfotransferase expression inducing an increased negative charge density of cartilage matrix. These negative charges might attract more water resulting in augmented compressive stiffness of the tissue. Therefore, decorin-deficient mice developed significantly less OA after forced exercise than WT mice.ConclusionsOur study demonstrates that the disruption of decorin-restricted TGFβ signalling leads to higher stiffness of articular cartilage matrix, rendering joints more resistant to OA. Therefore, the loss of an important structural component can improve cartilage homeostasis.

The cartilaginous growth plate is a well-organized and highly specialized tissue which drives the longitudinal elongation of bones developing through endochondral bone formation. A tightly regulated differentiation program of chondrocyte proliferation, maturation, and hypertrophy associated with extracellular matrix production, mineralization, and degradation culminates in the replacement of cartilage with bone. Chondrocytes within the growth plate are organized into anatomically well-defined horizontal zones, which reflect their morphologically, biochemically, and transcriptionally distinct differentiation stages. The differentiation process is accompanied by the establishment of cellular anisotropy and planar polarity that generates the unique spatial structure of the tissue. Proliferative chondrocytes acquire an elongated shape, align and divide orthogonally to the direction of the growth, and arrange into vertical columns that in most vertebrates direct the elongation process. Chondrocyte differentiation and polarity are essential and mutually interacting foundations of the normal growth pate function, and their disturbance results in chondrodysplasias with impaired longitudinal growth. This chapter will focus on the mechanisms responsible for the establishment and maintenance of the structural polarity of the cartilaginous growth plate.

Correct innervation of the main respiratory muscle in mammals, namely the thoracic diaphragm, is a crucial pre-requisite for the functionality of this muscle and the viability of the entire organism. Systemic impairment of Sema3A–Npn-1 (Npn-1 is also known as NRP1) signaling causes excessive branching of phrenic nerves in the diaphragm and into the central tendon region, where the majority of misguided axons innervate ectopic musculature. To elucidate whether these ectopic muscles are a result of misguidance of myoblast precursors due to the loss of Sema3A–Npn-1 signaling, we conditionally ablated Npn-1 in somatic motor neurons, which led to a similar phenotype of phrenic nerve defasciculation and, intriguingly, also formation of innervated ectopic muscles. We therefore hypothesize that ectopic myocyte fusion is caused by additional factors released by misprojecting growth cones. Slit2 and its Robo receptors are expressed by phrenic motor axons and migrating myoblasts, respectively, during innervation of the diaphragm. In vitro analyses revealed a chemoattractant effect of Slit2 on primary diaphragm myoblasts. Thus, we postulate that factors released by motor neuron growth cones have an influence on the migration properties of myoblasts during establishment of the diaphragm., Highlighted Article: Sema3A–Npn-1 signaling cell autonomously influences phrenic nerve branching in the diaphragm and induces ectopic muscle formation through secondary mechanisms, possibly by Slit–Robo signaling.

Fragestellung: Infolge der demographischen Entwicklung wird die Inzidenz osteoporoseassoziierter Frakturen in den nächsten Jahren ansteigen. Die proximale Femurfraktur ist eine der häufigsten und prognostisch schwerwiegendsten Manifestationen der Osteoporose. Patienten mit einer osteoporotischen[zum vollständigen Text gelangen Sie über die oben angegebene URL], Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2015)

Collagen IX (Col IX) is an important component of the cartilage extracellularmatrix and has been associated with degenerative cartilage disorders and chondrodysplasias in humans. Further, polymorphisms in Col IX are known risk factors for the development of early intervertebral disc (IVD) degeneration. To understand the role of Col IX in the pathogenesis of IVD disorders, the spine of newborn and older Col IX deficient mice was systematically analyzed and compared to C57BL/6N controls. Morphology and bone parameters of the spine from newborn, 6 and 10 months old animals were investigated using μCT measurements. Histological staining was used to evaluate tissue structure and degree of degeneration. Localization and expression of extracellularmatrix proteins was analyzed in depth by immunofluorescence staining, immunoblotting, RT-PCR and in situ hybridization. High resolution imaging and stiffness measurements were performed by atomic force microscopy (AFM). Vertebral bodies of newborn Col IX-deficient mice were smaller and showed an increased mineral density compared to wild type animals. At birth, lack of Col IX led to a disrupted cellular organization in the cartilaginous endplate and a smaller nucleus pulposus of the IVD.Expression levels and localization of other extracellularmatrix proteins were strongly altered accompanied by a softening of cartilaginous tissues. In older animals, absence of Col IX caused earlier and more pronounced disc degeneration with annular fissures. The absence of Col IX induces early developmental, structural and biomechanical alterations in both vertebral body and intervertebral disc which eventually cause severe degenerative changes in the aging spine.

A new diol-carbonyl transposition reaction has been discovered in echinocandin type structures. An α-hydroxy hemiaminal moiety has been shown to undergo a pinacol-type rearrangement in the presence of trimethylsilyl iodide to afford ketone derivatives. Applied to deoxymulundocandin, this transposition led to a useful intermediate for further chemical modification.

Due to the importance of the cephems, it is essential to classify them so as to allow their optimal use. The cephems are characterized by an azetidinone ring, to which is attached an unsaturated hexacycle. Five classes of cephems may be distinguished in terms of the nature of the moiety: cephalosporins, cephamycins and cephabacins, oxa-1-cephems, carba-1-cephems, and miscellaneous. The dosage and dosing frequency of a cephalosporin are the result of a balance between its antibacterial activity and its pharmacokinetic profile. The molecules all have the same antibacterial spectrum, which principally includes gram-negative bacilli and gram-negative and gram-positive cocci, except for S. aureus. The carbacephem-type molecules are asymmetrical, and the substituents on the β-lactam moiety are in the cis position; the cis stereochemistry of the carbacephems is less stable thermodynamically than the trans stereochemistry. The antibacterial activity RWJ-54428 was designed to overcome methicillin resistance in methicillin-resistant S. aureus (MRSA) strains and ampicillin resistance in E. faecium by penicillin-binding proteins (PBP) alterations. In all species, total clearance was slower and the apparent elimination half-life was longer than that of RWJ- 54428. The antistaphylococcal activities of RWJ-333442 in comparison with RWJ-54428, vancomycin, and quinupristindalfopristin against S. aureus Smith were investigated in a sepsis model (Swiss-Webster mice) and were also investigated for S. aureus 076, a methicillin-resistant strain. A synergistic activity was shown to be species dependent in combination with streptomycin against E. faecalis.