Hyaluronan Synthases' Expression and Activity Are Induced by Fluid Shear Stress in Bone Marrow-Derived Mesenchymal Stem Cells.

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.

Adhesive Properties of the Hyaluronan Pericellular Coat in Hyaluronan Synthases Overexpressing Mesenchymal Stem Cells.

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.

Chondrogenic Potential of Pellet Culture Compared to High-Density Culture on a Bacterial Cellulose Hydrogel.

Cell-based approaches of cartilage lesions use different culture systems to obtain optimal cell quality. Pellet cultures with high cellular density (HD) are the gold standard to keep chondrocytes in a differentiated stage. Bacterial cellulose (BC) hydrogel is discussed to prevent cellular aging and dedifferentiation. The hypothesis of this study was that HD culture on BC hydrogel (HD hydrogel) might reach the chondrogenic potential of pellet culture (pellet). Human articular osteoarthritic (OA) and non-osteoarthritic (non-OA) chondrocytes were cultured for seven days within pellets and compared to HD hydrogel and HD polystyrene. Gene expression analysis and histological assessment were performed. We observed no significant change of COL2A1 expression by the culture system (pellet, HD hydrogel and HD polystyrene) but a significant change of COL2A1/COL1A1-ratio, with the highest ratio in pellets. Chondrocytes on HD hydrogel showed an elevated expression of MMP13 and on polystyrene an increased expression of COL1A1 and MMP13. The patterns of gene expression changes observed in OA and non-OA chondrocytes in reaction to the different culture systems were similar in those two cell groups. Pellet cultures moreover formed a histomorphologically superior neocartilage. Concluding, human chondrocytes kept the potential to express COL2A1 in all HD culture systems. However, pellets excelled in a higher COL2A1/COL1A1-ratio, a higher extracellular matrix deposit and in not developing degeneration and dedifferentiation markers. This underlines the superiority of pellet culture in maintaining the chondrogenic potential of human chondrocytes in vitro.

First inducible transgene expression in porcine large animal models.

The purpose of this study was to establish inducible transgene expression in pigs, a model organism with great promise for experimental physiology and translational medicine, using the binary tet-on system. This expression system is activated by doxycycline (dox) via the tet-controlled transactivator (TA). Binding of TA to the transactivator response element (TRE) results in transcription of downstream genes. First, we cloned transgenic founder pigs expressing TA under the control of the CMV enhancer/chicken ß-actin promoter (CAG). Then, cells from CAG-TA transgenic founders were nucleofected with TRE-controlled expression vectors for either porcine cytotoxic T-lymphocyte associated antigen 4-Fc domain of immunoglobulin G1 (CTLA-4Ig) or soluble receptor activator of NF-κB ligand (RANKL), and double-transgenic offspring were cloned. Dox administration resulted in a dose-dependent increase in expression of CTLA-4Ig or RANKL, in nucleofected cells and in transgenic pigs, while in the absence of dox, the levels of both proteins were below the detection limit. Inducible transgene expression was reproduced in double-transgenic offspring generated by cloning or breeding. Our strategy revealed the first two examples of inducible transgene expression in pigs. The CAG-TA transgenic pigs generated in this study constitute an interesting basis for future pig models with inducible transgene expression.

Increased stemness and migration of human mesenchymal stem cells in hypoxia is associated with altered integrin expression.

Human mesenchymal stem cells (hMSCs) are regularly cultured and characterised under normoxic (21% O(2)) conditions, although the physiological oxygen tension in the stem cell niche is known to be as low as 1-2%. Oxygen itself is an important signalling molecule, but the distinct impact on various stem cell characteristics is still unclear. Therefore, the aim of this study was to evaluate the influence of oxygen concentration on the hMSC subpopulation composition, cell morphology and migration on different surfaces (polystyrene, collagen I, fibronectin, laminin) as well as on the expression of integrin receptors. Bone marrow-derived hMSCs were cultured either in normoxic (21% O(2)) or hypoxic (2% O(2)) conditions. The hMSC subpopulations were assessed by aspect ratio and cell area. Hypoxia promoted a more homogeneous cell population with a significantly higher fraction of rapidly self-renewing cells which are believed to be the true stem cells. Under hypoxic conditions hMSC volume and height were significantly decreased on all surfaces as measured by white light confocal microscopy. Furthermore, low oxygen tension led to a significant increase in cell velocity and Euclidian distance on all matrixes, which was evaluated by time-lapse microscopy. With regard to cell-matrix contacts, expression of several integrin subunits was evaluated by semi-quantitative RT-PCR. Increased expression of the subunits α(1), α(3), α(5,) α(6), α(11), α(v), ß(1) and ß(3) was observed in hypoxic conditions, while α(2) was higher expressed in normoxic cultured hMSCs. Taken together, our results indicate that hypoxic conditions promote stemness and migration of hMSC along with altering their integrin expression.

SFRP2 Overexpression Induces an Osteoblast-like Phenotype in Prostate Cancer Cells.

Prostate cancer bone metastasis is still one of the most fatal cancer diagnoses for men. Survival of the circulating prostate tumor cells and their adaptation strategy to survive in the bone niche is the key point to determining metastasis in early cancer stages. The promoter of SFRP2 gene, encoding a WNT signaling modulator, is hypermethylated in many cancer types including prostate cancer. Moreover, SFRP2 can positively regulate osteogenic differentiation in vitro and in vivo. Here, we showed SFRP2 overexpression in the prostate cancer cell line PC3 induces an epithelial mesenchymal transition (EMT), increases the attachment, and modifies the transcriptome towards an osteoblast-like phenotype (osteomimicry) in a collagen 1-dependent manner. Our data reflect a novel molecular mechanism concerning how metastasizing prostate cancer cells might increase their chance to survive within bone tissue.

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active.

Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown.

Extended in vitro culture of primary human mesenchymal stem cells downregulates Brca1-related genes and impairs DNA double-strand break recognition.

Mesenchymal stem cells (MSCs) are multilineage adult stem cells with considerable potential for cell-based regenerative therapies. In vitro expansion changes their epigenetic and cellular properties, with a poorly understood impact on DNA damage response (DDR) and genome stability. We report here results of a transcriptome-based pathway analysis of in vitro-expanded human bone marrow-derived mesenchymal stem cell (hBM-MSCs), supplemented with cellular assays focusing on DNA double-strand break (DSB) repair. Gene pathways affected by in vitro aging were mapped using gene ontology, KEGG, and GSEA, and were found to involve DNA repair, homologous recombination (HR), cell cycle control, and chromosomal replication. Assays for the recognition (γ-H2AX + 53BP1 foci) and repair (pBRCA1 + Î³-H2AX foci) of X-ray-induced DNA DSBs in hBM-MSCs show that over a period of 8 weeks of in vitro aging (i.e., about 10 doubling times), cells exhibit a reduced DDR and a higher fraction of residual DNA damage. Furthermore, a distinct subpopulation of cells with impaired DNA DSB recognition was observed. Several genes that participate in DNA repair by HR (e.g., Rad51, Rad54, BRCA1) show a 2.3- to fourfold reduction of their mRNA expression by qRT-PCR. We conclude that the in vitro expansion of hMSCs can lead to aging-related impairment of the recognition and repair of DNA breaks.

Correction to: Bone defect reconstruction with a novel biomaterial containing calcium phosphate and aluminum oxide reinforcement.

An amendment to this paper has been published and can be accessed via the original article.

Bone defect reconstruction with a novel biomaterial containing calcium phosphate and aluminum oxide reinforcement.

BACKGROUND: Reconstruction of metaphyseal fractures represents a clinical challenge for orthopedic surgeons. Especially in osteoporotic bone, these fractures are frequently accompanied by osseous substance defects. In order to ensure rapid mobilization of patients, high stability requirements must be met by osteosynthesis. Various bone graft materials have been introduced in the past, such as autologous bone or exogenous bone substitute materials. These are used as bone void fillers or as augmentation techniques to ensure safe fixation of osteosynthesis. New calcium phosphate-based bone void-filling materials could be a promising alternative to autologous bone or to the currently and widely used polymethylmethacrylate (PMMA)-based cement. The aim of this study was to evaluate a novel paste-like bone void filler in vivo and in vitro with regard to biocompatibility and osteoconductivity. METHODS: In addition to in vitro testing of cell compatibility using pre-osteoblasts (MC3T3-E1), 35 Wistar rats were treated in vivo with implantation of various material mixtures based on calcium phosphate and aluminum oxide reinforcement in a metaphyseal drill hole defect. After 4 weeks, an examination by micro-computed tomography (µCT) and histology was performed. RESULTS: The in vitro analysis showed good biocompatibility with a high cell survival of osteoblasts. In the in vivo experiments, a significantly higher bone ingrowth compared to the empty defect was shown by µCT and histological analysis. Here, the group receiving material reinforced with aluminum oxide (Al2O3) showed a bone volume/tissue volume (BV/TV) of 89.19% compared to a BV/TV of 83.14% for the empty defect (p = 0.0013). In the group treated with a polysaccharide matrix, no increase in BV/TV was observed given a mean ratio of 80.14%. Scoring of histological sections did not reveal a significant difference between CaP and CaP that was substituted with Al2O3. CONCLUSION: The results of this study show an encouraging first step towards the development of new pasty, bone void-filling materials. We demonstrated that a new paste-like bone-filling material, based on calcium phosphate granulates and aluminum oxide to provide strength, exhibits good biocompatibility and osteoconductivity. Further biomechanical test in an osteoporotic animal model will have to be performed, to prove feasibility in metaphyseal defects.

Fibroblast-like cells change gene expression of bone remodelling markers in transwell cultures.

INTRODUCTION: Periprosthetic fibroblast-like cells (PPFs) play an important role in aseptic loosening of arthroplasties. Various studies have examined PPF behavior in monolayer culture systems. However, the periprosthetic tissue is a three-dimensional (3D) mesh, which allows the cells to interact in a multidirectional way. The expression of bone remodeling markers of fibroblast-like cells in a multilayer environment changes significantly versus monolayer cultures without the addition of particles or cytokine stimulation. Gene expression of bone remodeling markers was therefore compared in fibroblast-like cells from different origins and dermal fibroblasts under transwell culture conditions versus monolayer cultures. METHODS: PPFs from periprosthetic tissues (n = 12), osteoarthritic (OA) synovial fibroblast-like cells (SFs) (n = 6), and dermal fibroblasts (DFs) were cultured in monolayer (density 5.5 × 103/cm2) or multilayer cultures (density 8.5 × 105/cm2) for 10 or 21 days. Cultures were examined via histology, TRAP staining, immunohistochemistry (anti-S100a4), and quantitative real-time PCR. RESULTS: Fibroblast-like cells (PPFs/SFs) and dermal fibroblasts significantly increased the expression of RANKL and significantly decreased the expression of ALP, COL1A1, and OPG in multilayer cultures. PPFs and SFs in multilayer cultures further showed a higher expression of cathepsin K, MMP-13, and TNF-α. In multilayer PPF cultures, the mRNA level of TRAP was also found to be significantly increased. CONCLUSION: The multilayer cultures are able to induce significant expression changes in fibroblast-like cells depending on the nature of cellular origin without the addition of any further stimulus. This system might be a useful tool to get more in vivo like results regarding fibroblast-like cell cultures.

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z.

BACKGROUND: Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. RESULTS: Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 microM - 10 microM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. CONCLUSION: Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types.

The chitin synthase involved in marine bivalve mollusk shell formation contains a myosin domain.

Chitin is a key component in mollusk nacre formation. However, the enzyme complex responsible for chitin deposition in the mollusk shell remained unknown. We cloned and characterized the chitin synthase of the marine bivalve mollusk Atrina rigida. We present here the first chitin synthase sequence from invertebrates containing an unconventional myosin motor head domain. We further show that a homologous gene for chitin synthase is expressed in the shell forming tissue of larval Mytilus galloprovincialis even in early embryonic stages. The new data presented here are the first clear-cut indication for a functional role of cytoskeletal forces in the precisely controlled mineral deposition process of mollusk shell biogenesis.

Sox2 is a potent inhibitor of osteogenic and adipogenic differentiation in human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are a promising target for cell-based bone regeneration. However, their application for clinical use is limited because hMSCs lose their ability for cell division and differentiation during longer in vitro cultivation. The osteogenic differentiation is regulated through a complex network of molecular signal transduction pathways where the canonical Wnt pathway plays an important role. Sox2, a known key factor for maintenance of cellular pluripotency in stem cells, is supposed to influence the Wnt pathway in osteoblasts. In this study, we overexpressed Sox2 in immortalized hMSCs by lentiviral gene transfer. Sox2 overexpression significantly reduced the osteogenic and adipogenic differentiation potentials. This effect was abolished by knockdown of Sox2 overexpression. In addition, Oct4 and Nanog, other key transcription factors for pluripotency, are strongly upregulated when Sox2 is overexpressed. Furthermore, Dkk1, a target gene of the Sox2-Oct4 heterodimer and a Wnt antagonist, is downregulated. Sox2 overexpression causes higher expression levels of ß-catenin, the central transcription factor of the canonical Wnt pathway. These results suggest that Sox2 keeps hMSCs in an undifferentiated state by influencing the canonical Wnt pathway. Regulated expression of Sox2 may be a promising tool to cultivate hMSCs in sufficient quantities for cell and gene therapy applications.

In vivo mesenchymal stem cell tracking with PET using the dopamine type 2 receptor and 18F-fallypride.

UNLABELLED: Human mesenchymal stem cells (hMSCs) represent a promising treatment approach for tissue repair and regeneration. However, little is known about the underlying mechanisms and the fate of the transplanted cells. The objective of the presented work was to determine the feasibility of PET imaging and in vivo monitoring after transplantation of dopamine type 2 receptor-expressing cells. METHODS: An hMSC line constitutively expressing a mutant of the dopamine type 2 receptor (D2R80A) was generated by lentiviral gene transfer. D2R80A messenger RNA expression was confirmed by reverse transcriptase-polymerase chain reaction. Localization of the transmembrane protein was analyzed by confocal fluorescence microscopy. The stem cell character of transduced hMSCs was investigated by adipogenic and osteogenic differentiation. Migration capacity was assessed by scratch assays in time-lapse imaging. In vitro specific binding of ligands was tested by fluorescence-activated cell sorting analysis and by radioligand assay using (18)F-fallypride. Imaging of D2R80A overexpressing hMSC transplanted into athymic rats was performed by PET using (18)F-fallypride. RESULTS: hMSCs showed long-term overexpression of D2R80A. As expected, the fluorescence signal suggested the primary localization of the protein in the membrane of the transduced cells. hMSC and D2R80A retained their stem cell character demonstrated by their osteogenic and adipogenic differentiation capacity and their proliferation and migration behavior. For in vitro hMSCs, at least 90% expressed the D2R80A transgene and hMSC-D2R80A showed specific binding of (18)F-fallypride. In vivo, a specific signal was detected at the transplantation site up to 7 d by PET. CONCLUSION: The mutant of the dopamine type 2 receptor (D2R80A) is a potent reporter to detect hMSCs by PET in vivo.

Molecular cloning and functional characterization of the porcine extracellular domain of Receptor Activator of NF-κB Ligand (sRANKL).

Receptor Activator of NF-κB Ligand (RANKL) plays a pivotal role as a regulator of osteoclast activity and is involved in osteoporosis. Here, we report the cloning and functional characterization of the complete extracellular domain of the porcine RANKL gene (sRANKL). The porcine sRANKL cDNA has an ORF of 744 nucleotides and shares 87%, 80% and 80% identity with human, rat and mouse RANKL coding sequences, respectively. The protein consists of 247 amino acids with 90%, 81% and 80% sequences similarities compared to human, mouse and rat RANKL, respectively. Over-expression of porcine sRANKL led to osteoclast formation. The osteoclasts showed a characteristic morphology, expressed the carbonic anhydrase type 2, were TRACP positive and exhibited a bone-resorbing activity. In conclusion, we first describe the molecular cloning and functional characterization of porcine sRANKL, which will help to understand the function of a RANKL gene in large animal models.

The distribution of chitin in larval shells of the bivalve mollusk Mytilus galloprovincialis.

The insoluble matrix of larval shells of the marine bivalve mollusk Mytilus galloprovincialis is investigated by confocal laser scanning microscopy using a GFP fusion protein with a chitin-binding domain for labeling of chitinous structures. We show that chitinous material is present in the larval shell, presumably as a chitin-protein complex. We further show that the structure of the chitinous material changes with the development of the larvae. We conclude from the presence of characteristic chitinous structures in certain shell regions that chitin fulfills an important function in the formation and functionality of larval bivalve shells.