Chiara Gentili

Cartilage and Bone Extracellular Matrix

The extracellular matrix (ECM) is a complex of self assembled macromolecules. It is composed predominantly of collagens, non-collagenous glycoproteins, hyaluronan and proteoglycans. ECM is not only a scaffold for the cells; it serves also as a reservoir for growth factors and cytokines and modulates the cell activation status and turnover. ECM should be considered a dynamic network of molecules secreted by cells that in turn regulate cell behavior by modulating their proliferation and differentiation. The ECM provides structural strength to tissues, maintaining a complex architecture around the cells and the shape of organs. Various cell types secrete different matrix molecules and the nature and the amount of these molecules change during developmental age. Cartilage ECM is composed mainly of two components defining its mechano-physical properties: the collagenous network, responsible for the tensile strength of the cartilage matrix, and the proteoglycans (mainly aggrecan), responsible for the osmotic swelling and the elastic properties of the cartilage tissue. The conversion of cartilage into bone requires several processes that directly involve the different ECM components. Homeostasis of cartilage and bone is maintained by complex mechanisms controlling turnover and remodeling of ECM. In bone, as well as in cartilage, the ECM resident cells produce local factors, inflammatory mediators, and matrix-degrading enzymes. Turnover and degradation of normal and pathological matrices are dependent on the responses of the local cell to auto and paracrine anabolic and catabolic pathway.

Amniotic liquid derived stem cells as reservoir of secreted angiogenic factors capable of stimulating neo-arteriogenesis in an ischemic model

Most urgent health problems are related to a blood vessel formation failure. The use of stem cells from different sources or species for both in vitro and in vivo engineering of endothelium does not necessarily imply their direct commitment towards a vascular phenotype. In the present study, we used human amniotic fluid stem cells (AFSC) to evoke a strong angiogenic response in murine recipients, in terms of host guided-regeneration of new vessels, and we demonstrated that the AFSC secretome is responsible for the vascularising properties of these cells. We indentified in AFSC conditioned media (ACM) pro-angiogenic soluble factors, such as MCP-1, IL-8, SDF-1, VEGF. Our in vitro results suggest that ACM are cytoprotective, pro-differentiative and chemoattractive for endothelial cells. We also tested ACM on a pre-clinical model of hind-limb ischemic mouse, concluding that ACM contain mediators that promote the neo-arteriogenesis, as remodelling of pre-existing collateral arteries to conductance vessels, thus preventing the capillary loss and the tissue necrosis of distal muscles. In line with the current regenerative medicine trend, in the present study we assert the concept that stem cell-secreted mediators can guide the tissue repair by stimulating or recruiting host reparative cells.

p38/NF-kB-dependent expression of COX-2 during differentiation and inflammatory response of chondrocytes.

Studying cartilage differentiation, we observed the emergence of inflammation‐related proteins suggesting that a common pathway was activated in cartilage differentiation and inflammation. In the present paper, we investigated the expression pathway of the inflammation‐related enzyme Cyclooxygenase‐2 (COX‐2) during differentiation and inflammatory response of the chondrocytic cell line MC615. Cells were cultured either as (i) proliferating prechondrogenic cells expressing type I collagen or (ii) differentiated hyperconfluent cells expressing Sox9 and type II collagen. The p38 and the NF‐kB pathways were investigated in standard conditions and after inflammatory agents treatment. NF‐kB was constitutively activated in differentiated cells. The activation level of NF‐kB in differentiated cells was comparable to the level in proliferating cells treated with the inflammatory agent LPS. In both cases, p65 was bound to the NF‐kB consensus sequence of COX‐2 promoter. p38, constitutively activated in differentiated cells, was activated in proliferating cells by treatment with LPS or IL‐1α. In stimulated proliferating cells the two pathways are connected since addition of the p38‐specific inhibitor SB203580 inhibited p38 activation, significantly reduced NF‐kB activation and repressed COX‐2 synthesis indicating that p38 is upstream NF‐kB activation and COX‐2 synthesis. In differentiated cells, the treatment with the inflammatory agent neither enhance NF‐kB activation, nor synthesis of COX‐2 while the addition of SB203580 neither repressed activation of p38, nor COX‐2 synthesis, suggesting a constitutive activation of a p38/NF‐kB/COX2 pathway. Our data indicate that in chondrocytes, COX‐2 is expressed via p38 activation/NF‐kB recruitment during both differentiation and inflammatory response. J. Cell. Biochem. 104: 1393–1406, 2008.

Novel injectable gel (system) as a vehicle for human articular chondrocytes in cartilage tissue regeneration

We developed a novel injectable carrageenan/fibrin/hyaluronic acid‐based hydrogel with in situ gelling properties to be seeded with chondrogenic cells and used for cartilage tissue engineering applications. We first analysed the distribution within the hydrogel construct and the phenotype of human articular chondrocytes (HACs) cultured for 3 weeks in vitro. We observed a statistically significant increase in the cell number during the first 2 weeks and maintenance of cell viability throughout the cell culture, together with the deposition/formation of a cartilage‐specific extracellular matrix (ECM). Taking advantage of a new in vivo model that allows the integration between newly formed and preexisting cartilage in immunodeficient mice to be investigated, we showed that injectable hydrogel seeded with human articular chondrocytes was able to regenerate and repair an experimentally made lesion in bovine articular cartilage, thus demonstrating the potential of this novel cell delivery system for cartilage tissue engineering. Copyright

Vis‐à‐Vis Cells and the Priming of Bone Formation

Bone formation throughout skeletal growth and remodeling always entails deposition of new bone onto a pre‐existing mineralized surface. In contrast, the initial deposition of bone in development requires the formation, ex novo, of the first mineralized structure in a nonmineralized tissue. We investigated the cellular events associated with this initial bone formation, with specific reference to the respective role of cartilage and bone cells in bones which form via a cartilage model. The cellular architecture of initial osteogenic sites was investigated by light, confocal, and electron microscopy (EM) in the membranous ossification of fetal calvarial bones (not forming via a cartilage model) and in the membranous ossification of the bony collars of endochondral bones. Bone sialoprotein (BSP), which is expressed during early phases of bone deposition and has been proposed to be involved in the control of both mineral formation and bone cell–matrix interactions, was used as a marker of initial bone formation. We found that at all sites, BSP‐producing cells (as identified by intracellular immunoreactivity) are arranged in a characteristic vis‐à‐vis (face to face) pattern prior to the appearance of the first mineralizing BSP‐immunoreactive extracellular matrix. In perichondral osteogenesis, the vis‐à‐vis pattern comprises osteoblasts differentiating from the perichondrium/periosteum and early hypertrophic chondrocytes located at the lateral aspects of the rudiment. By EM, the first mineral and the first BSP‐immunoreactive sites coincide temporally and spatially in the extracellular matrix at the boundary between cartilage and periosteum. We further showed that in an in vitro avian model of chondrocyte differentiation in vitro to osteoblast‐like cells, early hypertrophic chondrocytes replated as adherent cells turned on the expression of high levels of BSP in conjunction with the switch to collagen type I synthesis and matrix mineralization. We propose a model for the priming of bone deposition, i.e., the formation of the first bone structure, in which the architectural layout of cells competent to deposit a mineralizing matrix (the vis‐à‐vis pattern) determines the polarized deposition of bone. For bones forming via a cartilage model, the priming of bone deposition involves and requires cells that differentiate from early hypertrophic chondrocytes.

The Developmentally Regulated Avian Ch21 Lipocalin Is an Extracellular Fatty Acid-binding Protein

Ch21, a developmentally regulated extracellular protein expressed in chick embryos and in cultured chondrocytes, was expressed in the baculovirus system, and the recombinant protein was purified to homogeneity by gel-filtration chromatography. Separation of two isoforms was achieved on an ion-exchange column. Previous work had shown that Ch21 belongs to the superfamily of lipocalins, which are transport proteins for small hydrophobic molecules. Studies were performed to identify the Ch21 ligand. By analysis of recombinant Ch21 on native polyacrylamide gel electrophoresis and by Lipidex assay, the binding of fatty acid to the protein was shown and a preferential binding of long-chain unsaturated fatty acids was observed. Both isoforms had the same behavior. The binding was saturable. Stoichiometry was about 0.7 mol of ligand/mol of protein. The protein binds the ligand in its monomeric form. Calculated dissociation constants were 2 × 10−7 M for unsaturated fatty acids and 5 × 10−7 M for stearic acid. The binding was specific; other hydrophobic molecules, as retinoic acid, progesterone, prostaglandins, and long-chain alcohols and aldehydes did not bind to the protein. Short-chain fatty acids did not bind to the protein. Ch21, also present in chicken serum, represents the first extracellular protein able to selectively bind and transport fatty acid in extracellular fluids and serum. We propose to rename the Ch21 protein as extracellular fatty acid-binding rotein (Ex-FABP).

Proangiogenic soluble factors from amniotic fluid stem cells mediate the recruitment of endothelial progenitors in a model of ischemic fasciocutaneous flap.

Skin flaps are routinely used in surgery for the functional and cosmetic repair of wounds or disfiguring scars. The recent concept of therapeutic angiogenesis has emerged as an attractive approach to overcome the problem of blood supply deficiency, often resulting in the flap grafting failure. In the present study, we embedded a gelatin membrane with amniotic fluid stem cells (AFSC) derived conditioned media (ACM) to topically deliver angiogenic growth factors and cytokines into a rat model of ischemic full-thickness skin flap elevated in the epigastric region. AFSC secretome triggered the endogenous repair by the recruitment of endothelial progenitor cells. We studied the vascular perfusion rate, the vessel distribution, and the survival of ACM-treated flaps. In detail, the ischemic sectors of ACM-treated flaps showed at day 7 a perfusion level 50% higher than the preoperation baseline. The ensuing necrosis development was delayed and the histology analysis showed a normal arrangement of epidermal and dermal structures and a high density of vessels in subcutaneous tissues. Further, we found that ACM recruited CD31⁺/VEGFR2⁺ and CD31⁺/CD34⁺ cells into the ischemic subcutaneous tissues and that the isolated progenitors were capable to form clusters of von Willebrand factor-positive cells in culture. We propose ACM as a cell-free cocktail of chemokines and growth factors to be adopted for clinical applications.

The Regenerative Role of the Fetal and Adult Stem Cell Secretome

For a long time, the stem cell regenerative paradigm has been based on the assumption that progenitor cells play a critical role in tissue repair by means of their plasticity and differentiation potential. However, recent works suggest that the mechanism underlying the benefits of stem cell transplantation might relate to a paracrine modulatory effect rather than the replacement of affected cells at the site of injury. Therefore, mounting evidence that stem cells may act as a reservoir of trophic signals released to modulate the surrounding tissue has led to a paradigm shift in regenerative medicine. Attention has been shifted from analysis of the stem cell genome to understanding the stem cell “secretome”, which is represented by the growth factors, cytokines and chemokines produced through paracrine secretion. Insights into paracrine-mediated repair support a new approach in regenerative medicine and the isolation and administration of specific stem cell-derived paracrine factors may represent an extremely promising strategy, introducing paracrine-based therapy as a novel and feasible clinical application. In this review, we will discuss the regenerative potential of fetal and adult stem cells, with particular attention to their secretome.

Anti-inflammatory activity of monogalactosyldiacylglycerol in human articular cartilage in vitro: activation of an anti-inflammatory cyclooxygenase-2 (COX-2) pathway

IntroductionThe mono- and digalactosyldiacylglycerol (MGDG and DGDG) galactolipids have been purified from the thermophilic blue-green alga Phormidium sp. ETS-05 that colonizes the therapeutic thermal mud of Abano Terme and Montegrotto Terme, Italy. Both compounds present a marked composition in polyunsaturated fatty acids, mainly omega-3. The therapeutic thermal mud is applied mainly to osteoarthritic cartilage patients. In the present study the effect of MGDG treatment on proteins and factors expressed by human articular cartilage cells in culture and on pathways activated in inflammatory conditions was studied.MethodsPrimary cultures of human articular chondrocytes were used at cell passage number 1 (P1). Cells were treated in serum-free medium with inflammatory cytokines in the presence and in the absence of MGDG. Western blot was performed on collected medium and on cell layers. At least three different experiments were performed on primary cultures. The quantitation of the MGDG effect was performed by densitometric scanning of Western blots. p38 Mitogen Activated Protein Kinase (p38) activation, Nuclear Factor-kappaB (NF-kB) activation and Prostaglandin E2 (PGE2) quantitation were performed by commercially available assays. Results are given as the mean values ± SD. All statistical analyses were performed using GraphPad software. The two-tailed Students t -test was performed.ResultsWe report that MGDG: 1) represses the expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) induced by interleukin-1alpha (IL-1α) or IL-1α + tumor necrosis factor α (TNFα) interfering with the p38 and NF-kB pathways; 2) is not toxic for the cells and does not affect the cell phenotype; 3) strongly enhances COX-2 expression induced by IL-1α or IL-1α + TNFα; 4) represses mPGES expression induced by IL-1α and the synthesis of PGE2 and induces the synthesis of 15-deoxy-Δ 12,14-prostaglandin J2 (15ΔPGJ2). In addition, the COX-2 product 15ΔPGJ2 added to the cells: 1) strongly represses IL-6 and IL-8 induced by IL-1α; 2) represses mPGES expression induced by IL-1α and the synthesis of PGE2.ConclusionsAll together these data suggest that MGDG has an anti-inflammatory activity in human articular cartilage and possibly activates an anti-inflammatory loop triggered by COX-2 via 15ΔPGJ2 production, indicating a possible role of COX-2 in resolution of inflammation. The purified compound is a novel anti-inflammatory agent potentially active for human articular cartilage pathologies related to inflammation.

Monogalactosyldiacylglycerol anti-inflammatory activity on adult articular cartilage.

The mono- and the digalactosyldiacylglycerol (MGDG and DGDG) galactolipids with a high content of polyunsaturated fatty acids, mainly omega-3, have been purified from the thermophilic blue-green alga ETS-05 that colonises the therapeutic thermal mud of Abano and Montegrotto, Italy. The therapeutic thermal mud is applied mostly to osteoarthritic cartilage patients. In the present study, a possible anti-inflammatory function of MGDG in cartilage has been studied in the avian articular cartilage model. We report that, in response to an inflammatory stimulus, adult avian articular cartilage cells express inflammation-related proteins, such as the lipocalin extracellular fatty acid binding protein, Avidin and Serum Amyloid A. The treatment of avian articular chondrocytes with the galactolipid MGDG suppressed the expression of the inflammation-induced proteins, suggesting a strong anti-inflammatory property of MGDG. MGDG has, in addition, a cell anti-proliferative activity, but it does not interfere with cell differentiation, suggesting a protective role for articular cartilage.