Search

Damage of hyaline cartilage such as nasoseptal cartilage requires proper reconstruction, which remains challenging due to its low intrinsic repair capacity. Implantation of autologous chondrocytes in combination with a biomimetic biomaterial represents a promising strategy to support cartilage repair. Despite so far mostly tested for bone tissue engineering, bioactive glass (BG) could exert stimulatory effects on chondrogenesis. The aim of this work was to produce and characterize composite porous poly(L-lactide) (PLLA)/1393BG scaffolds via thermally induced phase separation (TIPS) technique and assess their effects on chondrogenesis of nasoseptal chondrocytes. The PLLA scaffolds without or with 1, 2.5, 5% BG1393 were prepared via TIPS technique starting from a ternary solution (polymer/solvent/non-solvent) in a single step. Scaffolds were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and differential scanning calorimetric analysis (DSC). Human nasoseptal chondrocytes were seeded on the scaffolds with 1 and 2.5% BG for 7 and 14days and cell survival, attachment, morphology and expression of SOX9 and cartilage-specific extracellular cartilage matrix (ECM) components were monitored. The majority of chondrocytes survived on all PLLA scaffolds functionalized with BG for the whole culture period. Also inner parts of the scaffold were colonized by chondrocytes synthesizing an ECM which contained glycosaminoglycans. Type II collagen and aggrecan gene expression increased significantly in 1% BG scaffolds during the culture. Chondrocyte protein expression for cartilage ECM proteins indicated that the chondrocytes maintained their differentiated phenotype in the scaffolds. BG could serve as a cytocompatible basis for future scaffold composites for osteochondral cartilage defect repair. Abbreviations: AB: alcian blue ACAN: gene coding for aggrecan; BG: Bioactive glass; 2D: two-dimensional; 3D: three-dimensional; COL2A1: gene coding for type II collagen; DAPI: 4ʹ,6-diamidino-2-phenylindole; DMEM: Dulbecco’s Modified Eagle’s Medium; DMMB: dimethylmethylene blue; DSC: Differential scanning calorimetric analysis; ECM: extracellular matrix; EDTA: ethylenediaminetetraacetic acid; EtBr: ethidium bromide; FCS: fetal calf serum; FDA: fluorescein diacetate; GAG: glycosaminoglycans; HDPE: high density polyethylene; HE: hematoxylin and eosin staining; HCA: hydoxylapatite; PBE: phosphate buffered EDTA100mM Na2HPO4 and 5mM EDTA, pH8; PBS: phosphate buffered saline; PFA: paraformaldehyde; PG: proteoglycans; PI: propidium iodide; PLLA: Poly-L-Lactic Acid Scaffold; RT: room temperature; SD: standard deviation; SEM: scanning electron microscopy; sGAG: sulfated glycosaminoglycans; SOX9/Sox9: SRY (sex-determining region Y)-box 9 protein; TBS: TRIS buffered saline; TIPS: Thermally Induced Phase Separation; XRD: X-ray diffraction analysis.

Butyric acid (BA) affects the differentiation of mesenchymal stem cells (MSC) through the activation of different transcriptional pathways. The aim of this study was to determine the effects of BA on proliferation and spontaneous differentiation of porcine bone marrow–derived MSC. Second passage MSC (n = 6) were cultured in either a basal medium (BM, DMEM + 10% FBS), or BM + 2.5 mmol/L BA (BA-2.5) or BM + 5 mmol/L BA (BA-5). Cell proliferation was significantly decreased by both BA-2.5 and BA-5 after 48 h and 72 h (− 55% and − 63%, respectively). To assess the impact of BA on spontaneous differentiation, MSC were cultured for 27 d, with complete media changes every 3 d. At day 27, cells were stained for osteocytic, chondrocytic, and adipocytic differentiation. No terminal differentiation was detected in control MSC, while accumulated small drops of lipids were stained by Oil-Red-O in BA-treated cells. The phenotypic changes were associated with changes in gene expression, determined by qPCR. Treatment with BA modulated the expression of adipocytic differentiation markers: peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α were significantly increased by both BA-2.5 and BA-5 throughout the study, while lipoprotein lipase and fatty acid-binding protein 4 were increased by BA-5 at day 3, and decreased by both BA-5 and BA-2.5 later throughout the study. Osteocalcin and aggrecan mRNA was reduced throughout the experiment by both doses of BA (P

Mesenchymal stem cells, a subpopulation of mesenchymal stromal cells (MSCs), are present in the stroma of several tissues. MSC in vitro cultivation for clinical treatments may greatly affect MSC properties. A primary handicap is replicative senescence that impairs MSC functions. Hyaluronan (HA) is present in the extracellular matrix that composes the stem cell niche environment and is under investigation as a key factor for in vitro stem cell growth. We evaluated the effect on MSC cultivation of HA hybrid cooperative complexes (HCC) that are obtained from high (H) and low (L) weight molecules (NAHYCO™). We compared this HCC with H-HA and L-HA. We investigated the effects of these HAs on proliferation, cell cycle, apoptosis, senescence, and differentiation following the addition of the polymer solutions in the culture media at concentrations that did not drastically modify the medium viscosity. Interestingly, 0,16% HCC significantly delayed the senescence compared with the controls. This occurred without alteration of the cell cycle, cytotoxicity, or apoptosis. HCCs also promoted adipogenic and chondrogenic differentiation. Our finding could suggest a potential functional role of HCC above the updated scientific reports of its effects and pave the way to optimization of MSC cultivation for therapeutic application.

Fractures heal predominantly through the process of endochondral ossification. The classic model of endochondral ossification holds that chondrocytes mature to hypertrophy, undergo apoptosis and new bone forms by invading osteoprogenitors. However, recent data demonstrate that chondrocytes transdifferentiate to osteoblasts in the growth plate and during regeneration, yet the mechanism(s) regulating this process remain unknown. Here, we show a spatially-dependent phenotypic overlap between hypertrophic chondrocytes and osteoblasts at the chondro-osseous border in the fracture callus, in a region we define as the transition zone (TZ). Hypertrophic chondrocytes in the TZ activate expression of the pluripotency factors [Sox2, Oct4 (Pou5f1), Nanog], and conditional knock-out of Sox2 during fracture healing results in reduction of the fracture callus and a delay in conversion of cartilage to bone. The signal(s) triggering expression of the pluripotency genes are unknown, but we demonstrate that endothelial cell conditioned medium upregulates these genes in ex vivo fracture cultures, supporting histological evidence that transdifferentiation occurs adjacent to the vasculature. Elucidating the cellular and molecular mechanisms underlying fracture repair is important for understanding why some fractures fail to heal and for developing novel therapeutic interventions.

Spray-dried microparticles of a derivative of hyaluronic acid (HA) have been engineered to obtain a controlled aggregation with Human Mesenchymal Stem Cells (hMSCs) into 3D constructs. We demonstrated the utility of chemical functionalization of a native constituent of the extracellular matrix to improve processing performances and to control on stem cell adhesion and differentiation. Native hyaluronic acid (HA), cell adhesive peptides (RGD), transforming growth factor β3, dexamethasone are biological agents potentially suitable for chondrogenic stimulation of hMSCS. However unmodified HA suffers of drawbacks in terms of stability and versatility of processing. Functionalization strategies are needed to overcome these drawbacks. In this paper microparticles were produced by spray-drying of an aliphatic and amino functionalized HA derivative. Hydrophobic derivatization of HA allowed the production of microparticles stabilized by physical crosslinking and to load and to control dexamethasone release. The presence of pendant amino groups was exploited to tether cyclic RGD and transforming growth factor β3 via maleimide chemistry; cyRGDC functionalization controlled hMSCs/microparticles aggregation. Chondrogenic potential was preliminary assayed by qualitative immunohistological detection.

3?-Sialyllactose and 6?-sialyllactose have been covalently linked to collagen films. Preliminary in vitro study on the behavior of mesenchymal stem cells (MSCs) in terms of cell viability, proliferation and induction of osteogenic and chondrogenic related genes has been performed. Results indicate that sialoside epitopes on collagen surface represent a suitable support for MSCs adhesion and cell proliferation, moreover, the neoglycosylation provide MSCs with different and specific stimuli, saccharide-type depending, in term of expression of osteogenic and chondrogenic related genes. In particular, 3?-sialyllactose significantly upregulate the expression of RUNX2 and ALP, well-known markers of osteogenesis, whereas 6?-sialyllactose up-regulate the expression of chondrocyte marker ACAN. Because no osteogenic or chondrogenic supplements in culture media were added, the inductive effect in terms of increased gene expression has to be ascribed uniquely to collagen surface functionalization. These results support the promising role of sialosides in the regulation of stem cells fate and open brilliant perspective for the future use of the presented approach toward osteochondral tissue engineering applications.

Chlorpyrifos (CPF) is an organophosphate insecticide used primarily to control foliage and soil-borne insect pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce dose-related abnormalities such as slower brain growth and cerebral cortex thinning. In lower vertebrates, for example, fish and amphibians, teratogenic activity of this compound is correlated with several anatomical alterations. Little is known about the effects of CPF on mRNA expression of genes involved in early development of the anatomical structures appearing abnormal in embryos. This study investigated the effects of exposure to different CPF concentrations (10, 15 and 20 mg/L) on Xenopus laevis embryos from stage 4/8 to stage 46. Some of the morphological changes we detected in CPF-exposed embryos included cranial neural crest cell (NCC)-derived structures. For this reason, we analyzed the expression of select genes involved in hindbrain patterning (egr2), cranial neural crest chondrogenesis, and craniofacial development (fgf8, bmp4, sox9, hoxa2 and hoxb2). We found that CPF exposure induced a reduction in transcription of all the genes involved in NCC-dependent chondrogenesis, with largest reductions in fgf8 and sox9; whereas, in hindbrain, we did not find any alterations in egr2 expression. Changes in the expression of fgf8, bmp4, and sox9, which are master regulators of several developmental pathways, have important implications. If these changes are confirmed to belong to a general pattern of alterations in vertebrates prenatally exposed to OP, they might be useful to assess damage during vertebrate embryo development. Environ. Mol. Mutagen., 2016. © 2016 Wiley Periodicals, Inc.

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo. Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

Matrix metalloproteinases (MMPs), known as matrixins, are Ca- and Zn-dependent endoproteinases involved in a wide variety of developmental and disease-associated processes, proving to be crucial protagonists in many physiological and pathological mechanisms. The ability of MMPs to alter, by limited proteolysis and through the fine control of tissue inhibitors of metalloproteinases, the activity or function of numerous proteins, enzymes, and receptors suggests that they are also involved in various important cellular functions during development. In this review, we focus on the differentiation of mesenchymal stem cells (including those of the myoblastic, osteoblastic, chondroblastic, neural, and apidoblastic lineages) and the possible, if unexpected, biological significance of MMPs in its regulation. The MMP system has been implicated in several differentiation events that suggests that it mediates the proliferative and prodifferentiating effect of the matrixin proteolytic cascade. We summarize these regulatory effects of MMPs on the differentiation of mesenchymal stem cells and hypothesize on the function of MMPs in the stem cell differentiation processes.

The transcription factor SOX9 is believed to be the master regulator of chondrogenesis. SOX8 is another SOX group E transcription factor with a high degree of homology to SOX9. Here, we demonstrate that SOX8 mRNA levels decrease during in vitro dedifferentiation of human articular chondrocytes and increase during chondrogenic differentiation of mesenchymal stromal cells. Knockdown of SOX9 reduced the expression of SOX8, COL2A1, and a range of other chondrogenic molecules. SOX8 knockdown reduced the expression of a large number of overlapping chondrogenic molecules, but not SOX9. Neither siSOX9 nor siSOX8 altered expression of the hypertrophic marker gene COL10A1. siSOX9, but not siSOX8 led to upregulation of hypertrophy associated genes MMP13 and ALPL. Transfection of synthetic SOX5, 6, and 9 mRNA trio upregulated SOX8, COL2A1, and ACAN, but not COL10A1 mRNA. Replacement of synthetic SOX9 by SOX8 in the SOX trio showed similar but lower chondrogenic effect. We conclude that SOX8 expression is regulated by SOX9, and that both together with SOX5 and SOX6 are required as a SOX quartet for transcription of COL2A1 and a large number of other chondrogenic molecules. Neither SOX8 nor SOX9 affect COL10A1 expression, but SOX9 inhibits chondrocyte hypertrophy through inhibition of MMP13 and ALPL expression. Stem Cells 2014;32:1629–1635

A Tight Squeeze During intracellular transport, the export of procollagen from the endoplasmic reticulum is intriguing because procollagen is too large to fit into conventional coat protein complex II (COPII)–coated transport vesicles. Recent work has implicated the receptor TANGO1 in procollagen export. Now, Venditti et al. (p. 1668 ) report that TANGO1 recruits Sedlin—also known as TRAPPC2, a homolog of the yeast TRAPP subunit Trs20—and helps to allow COPII-coated carriers to grow large enough to incorporate procollagen.