Search

Cell-based therapies using adipose-derived mesenchymal stromal cells (ADMSCs) have shown promising results for the treatment of osteoarthritis (OA). In fact, ADMSCs are now indicated as one of the most powerful cell sources through their immunomodulatory and anti-inflammatory activities. Recently, an innovative one-step closed device was developed to obtain microfragmented adipose tissue (MF) to avoid the need for good manufacturing practices for ADMSCs expansion while maintaining their regenerative potential. The aim of this study was to assess the mechanisms of action of MF and ADMSCs from MF (MF-ADMSCs) on an inflammatory cell model of OA synoviocytes. We found that MF produced low levels of inflammatory factors such as interleukin 6 (IL-6), CC-chemokine ligand 5/receptor-activated normal T-cell expressed and secreted (CCL5/RANTES), CC-chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1), and CC-chemokine ligand 3/macrophage inflammatory protein-1α (CCL3/MIP-1α), and a higher level only of CXC-chemokine ligand 8/interleukin 8 compared with MF-ADMSCs. Matrix metalloproteinase 9 (MMP-9) degradative factor but released a lower level of its inhibitor tissue inhibitor of the metalloproteinase (TIMP-1). MF in coculture with synoviocytes significantly induced both the metabolic activity and the release of IL-6. In contrast, MF, not MF-ADMSCs, partially decreased CCL5/RANTES. Moreover, MF reduced the release of both macrophage-specific chemokines (CCL2/MCP-1 and CCL3/MIP-1α) and degradative marker MMP-9. Interestingly, MF increased TIMP-1 (the MMP-9 inhibitor) and down-modulated toll-like receptor (TLR4) receptor and key molecules of NFκB pathways. These data evidenced different effects of MF versus MF-ADMSCs on inflamed synoviocytes. MF reduced typical macrophages markers and its potentiality by switching off macrophages activity was strictly dependent on TLR4 and NFκB signaling., (© 2018 Wiley Periodicals, Inc.)

Mesenchymal stromal cells (MSCs) are key players in the repair or regeneration of the damaged bone tissue. However, heterogeneity exists between MSCs derived from different donors in their bone formation ability both in vitro and in vivo. The identification of markers defining MSCs with different functional phenotypes is fundamental to maximize their clinical potential. In our previous in vivo study, impaired expression in MSCs of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), the two key enzymes in the catabolic pathway of homocysteine, was associated to decreased bone formation and to the onset of osteoporosis in mice. Here, we investigated whether osteogenic differentiation of human MSCs (hMSCs) modulates the expression of CBS and CSE. The expression of CBS and CSE was also assessed during chondrogenesis to confirm the specificity of their expression during osteogenesis. hMSCs displayed a heterogeneous mineralizing capacity between donors (70% of the samples mineralized, while 30% did not mineralize). Inducible expression of CBS and CSE was found to be associated with a mineralizing phenotype in hMSCs. In particular, up-regulation of CSE was restricted to hMSCs undergoing mineralization. During chondrogenesis, CBS was significantly up-regulated while CSE expression was not affected. Ex-vivo findings confirmed that mature h-osteoblasts (hOBs) show consistently higher expression of CBS and CSE than hMSCs. Our data provide the first evidence that the expression of CBS and CSE in hMSCs closely correlates with the transition of hMSCs toward the osteoblastic phenotype and that CSE may constitute a novel marker of osteogenic differentiation., (© 2017 Wiley Periodicals, Inc.)

Fluctuation in extracellular calcium (Ca(2+)) concentration occurs during bone remodeling. Free ionized Ca(2+) plays a critical role in regulating osteoblast functions. We analyzed the effects of different concentrations of free ionized Ca(2+) (0.5, 1.3, and 2.6 mM) on human osteoblasts and we evaluated osteoblastic phenotype (marker expression and cell morphology) and functions (osteogenic differentiation, cell proliferation, and cell signaling). Our data show human osteoblasts that chronically stimulated with 0.5, 1.3, or 2.6 mM Ca(2+) significantly increase intracellular content of alkaline phosphatase, collagen type I, osteocalcin, and bone sialoprotein, whereas collagen type XV was down-modulated and RUNX2 expression was not affected. We also found a Ca(2+) concentration-dependent increase in osteogenic differentiation and cell proliferation, associated to an increase of signaling protein PLCβ1 and p-ERK. Human osteoblast morphology was affected by Ca(2+) as seen by the presence of numerous nucleoli, cells in mitosis, cell junctions, and an increased number of vacuoles. In conclusion, our data show a clear phenotypical and functional effect of extracellular Ca(2+) on human osteoblasts and support the hypothesis of a direct role of this cation in the bone remodeling processes., (Copyright © 2011 Wiley Periodicals, Inc.)

Human bone cells used for in vitro studies are mainly derived from bone marrow (BM) or trabecular bone (TB). There are no specific markers or procedures for isolation and growth of these cells. To validate the potentiality of these cells, we isolated human mesenchymal stromal cells (MSCs) and osteoblasts (OBs) from the tibial plateau of the same subject, grown in two different media (α-MEM and DMEM/F12) and analyzed for cell growth, proliferation, phenotype and osteogenic potential. We found that OBs grew well in both media tested, but MSCs were able to grow only in α-MEM medium. OBs in DMEM/F12 showed reduced proliferation capability and expressed a low level of alkaline phosphatase (AP), RUNX-2, osteocalcin (OC), bone sialoprotein (BSP), collagen type I (Col.I) compared with OBs in α-MEM but high level of collagen type XV (Col.XV). Compared with MSCs in α-MEM, OBs have an increased ability to proliferate and express more OC and BSP at molecular level but less AP, RUNX-2 and Col.I than MSCs. Time-course experiments to analyze the osteogenic potential of these cells showed that OBs were more efficient than MSCs. However, these cells obtained from tibial plateau showed a different trend of AP, OC and Col.I osteogenic markers compared to control MSCs from the iliac crest. This study shows that bone-adherent OBs grown in α-MEM medium are more efficient for osteogenic differentiation than BM MSCs and contribute to defining their phenotypic and functional characteristics, so providing a rationale for their use in bone tissue engineering or therapeutic purposes., (Copyright © 2011 Wiley-Liss, Inc.)

With the continuous discovery of new alternative sources containing mesenchymal stem cells (MSCs), regenerative medicine therapies may find tailored applications in the clinics. Although these cells have been demonstrated to express specific mesenchymal markers and are able to differentiate into mesenchymal lineages in ad hoc culture conditions, it is still critical to determine the yield and differentiation potential of these cells in comparative studies under the same standardized culture environment. Moreover, the opportunity to use MSCs from bone marrow (BM) of multiorgan donors for cell banking is of relevant importance. In the attempt to establish the relative potential of alternative MSCs sources, we analyzed and compared the yield and differentiation potential of human MSCs from adipose and BM tissues of cadaveric origins, and from fetal annexes (placenta and umbilical cord) after delivery using standardized isolation and culture protocols. BM contained a significantly higher amount of mononuclear cells (MNCs) compared to the other tissue sources. Nonetheless, a higher cell seeding density was needed for these cells to successfully isolate MSCs. The MNCs populations were highly heterogeneous and expressed variable MSCs markers with a large variation from donor to donor. After MSCs selection through tissue culture plastic adhesion, cells displayed a comparable proliferation capacity with distinct colony morphologies and were positive for a pool of typical MSCs markers. In vitro differentiation assays showed a higher osteogenic differentiation capacity of adipose tissue and BM MSCs, and a higher chondrogenic differentiation capacity of BM MSCs., (Copyright © 2011 Wiley-Liss, Inc.)

T cells are critical regulators of osteoclast differentiation and function in bone, but whether osteoclasts can, in turn, regulate T cell homing, and response to stimuli is unclear. To investigate whether osteoclasts are immune competent cells, the expression of HLA Class II and costimulatory receptors was evaluated by RT-PCR and immunohistochemistry by comparing osteoclast precursors and mature osteoclasts. T-cell-attracting chemokines were measured in the supernatants of confluent cultures of osteoclasts and compared with mesenchymal stromal cells and osteoblasts. T cell proliferation, cytokine production, and apoptosis were assayed in co-cultures with osteoclasts in the presence or absence of mitogenic stimuli. To define the mechanism of action of osteoclasts, cytokine-blocking experiments were performed. Our findings revealed that mature osteoclasts constitutively expressed Class II HLA in the membrane and upregulate the expression of CD40 and CD80 during differentiation. Osteoclasts secreted high levels of most T cell chemoattractants and effectively retained T cells in adhesion assays. Moreover, the osteoclasts potently blunted T cell response to PHA and CD3/CD28 stimulation, thus inhibiting proliferation, suppressing T cell TNFα and IFNγ production and decreasing T cell apoptosis by a mostly cell-contact independent mechanism. In conclusion, osteoclasts are immune-competent cells which can retain T cells and suppress in vitro T cell response to proliferative stimuli., (Copyright © 2010 Wiley-Liss, Inc.)

Subchondral bone remodeling in osteoarthritis (OA) and rheumatoid arthritis (RA) is mainly characterized by the formation of osteophytes/fibrosis and by the presence of infiltrating cells associated to bone resorption. In this study we analyzed CC (cysteine cysteine motif) chemokine ligand (CCL)20 and CC chemokine receptor (CCR)6 function in subchondral bone tissue and osteoblasts isolated from OA and RA patients. CCL20/CCR6 expression was evaluated by immunohistochemical techniques in bone tissue from OA and RA patients. CCL20-functional tests were performed on osteoblasts isolated from OA and RA patients to evaluate enzymatic response and cell proliferation. Moreover, we assessed Akt phosphorylation as the major signaling pathway for CCL20. In bone tissue biopsies we found that osteoblasts from both OA and RA patients expressed CCR6 while CCL20 was expressed only by RA osteoblasts. Both CCR6 and CCL20 were highly expressed in osteocytes and mononuclear cells from only RA patients. CCL20-stimulated OA osteoblasts showed a significant increase in beta-N-acetylhexosaminidase release compared to RA. Conversely, a significant increase in cellular proliferation was found only in CCL20-stimulated RA osteoblasts associated to Akt phosphorylation. These data were confirmed in bone tissue biopsies. This study demonstrates a different expression of CCL20-positive osteoblasts in OA versus RA disease that seem to be associated with the presence of infiltrating mononuclear cells. Moreover, CCL20 stimulation resulted in a greater proliferative response in RA osteoblasts compared to OA osteoblasts, mediated by Akt signaling, while OA osteoblasts showed increased enzymatic activity, thus suggesting a differential role of this chemokine in OA and RA., (Copyright 2009 Wiley-Liss, Inc.)

Bone marrow stromal cells (MSCs) and osteoblasts are the two main non-haematopoietic cellular components of human bone tissue. To identify novel osteoblast-related molecules, we performed a gene expression profiling analysis comparing MSCs and osteoblasts isolated from the same donors. Genes differentially overexpressed in osteoblasts were mainly related to the negative control of cell proliferation, pro-apoptotic processes, protein metabolism and bone remodelling. Notably, we also identified the collagen XV (COL15A1) gene as the most up-regulated gene in osteoblasts compared with MSCs, previously described as being expressed in the basement membrane in other cell types. The expression of collagen type XV was confirmed at the protein level on isolated osteoblasts and we demonstrated that it significantly increases during the osteogenic differentiation of MSCs in vitro and that free ionised extracellular calcium significantly down-modulates its expression. Moreover, light and electron microscopy showed that collagen type XV is expressed in bone tissue biopsies mainly by working osteoblasts forming new bone tissue or lining bone trabeculae. To our knowledge, these data represent the first evidence of the expression of collagen type XV in human osteoblasts, a calcium-regulated protein which correlates to a specific functional state of these cells.

Protein kinase C (PKC) is a family of enzymes detected in a diverse range of cell types where they regulate various cellular functions such as proliferation, differentiation, cytoskeletal remodelling, cytokine production, and receptor-mediated signal transduction. In this study we have analyzed the expression of 11 PKC isoforms (-alpha, -beta(I), -beta(II), -gamma, -delta, -eta, -theta, -epsilon, -zeta, -iota/lambda, and -micro) in osteoblasts from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) in comparison with osteoblasts from post-traumatic (PT) patients. By Western blotting analysis, nine isoforms, -alpha, -beta(I), -beta(II), -delta, -theta, - epsilon, -zeta, - iota/lambda, and -micro, were detected in osteoblasts. In RA and OA patients, PKC -theta and -micro were greater expressed whereas PKC-epsilon and -zeta decreased when compared with normal cells. The subcellular distribution and quantitative differences were confirmed by immuno-electron microscopy. Furthermore, we demonstrated that treatment with the proinflammatory cytokines, IL-1beta and TNF-alpha, significantly decreased PKC-zeta expression in PT osteoblasts. This suggests that proinflammatory cytokines can modulate the expression of this PKC isoform in osteoblasts in a way which is similar to changes detected in arthritic patients., ((c) 2007 Wiley-Liss, Inc.)

We evaluated the role of CCL20 (MIP-3alpha) chemokine in cells directly involved in the remodeling of bone tissue (osteoblasts and osteoclasts) and we confirmed its expression in the subchondral bone tissue of rheumatoid arthritis (RA) patients. The expression of CCL20 and of its receptor CCR6 was evaluated in osteoblasts isolated from bone tissue of post-traumatic (PT) patients. Functional tests were performed to evaluate osteoblast proliferation and matrix protein modulation. Immunohistochemical analysis for CCR6, CCL20, and RANKL was performed on bone samples from RA patients. The role of CCL20 was then analyzed in osteoclast differentiation. We found that in basal conditions CCR6, but not its ligand CCL20, was highly expressed by osteoblasts. Functional analysis on osteoblasts showed that CCL20 significantly increased cellular proliferation but did not affect matrix protein expression. Pro-inflammatory cytokines significantly induced the release of CCL20 and RANKL by human osteoblasts but did not modulate CCR6 expression. Increased expression of CCR6, CCL20, and RANKL was confirmed in RA subchondral bone tissue biopsies. We demonstrated that CCL20 was also an earlier inducer of osteoclast differentiation by increasing the number of pre-osteoclasts, thus favoring cell fusion and MMP-9 release. Our results add new insight to the important role of the CCL20/CCR6, RANKL system in the bone tissue of RA. The contemporary action of CCL20 on osteoblasts and osteoclasts involved in the maintenance of bone tissue homeostasis demonstrates the important role of this compartment in the evolution of RA, by showing a clear uncoupling between new bone formation and bone resorption., (Copyright 2006 Wiley-Liss, Inc.)

Hyaluronan (HA), in the bone marrow stroma, is the major non-protein glycosaminoglycan component of extracellular matrix (ECM) involved in cell positioning, proliferation, differentiation as well as in receptor-mediated changes in gene expression. Repair of bone and regeneration of bone marrow is dependent on ECM, inflammatory factors, like chemokines and degradative factors, like metalloproteinases. We analyzed the interaction between human mesenchymal stem cells (h-MSCs) and a three-dimensional (3-D) HA-based scaffold in vitro. The expression of CXC chemokines/receptors, CXCL8 (IL-8)/CXCR1-2, CXCL10 (IP-10)/CXCR3, CXCL12 (SDF-1)/CXCR4, and CXCL13 (BCA-1)/CXCR5, and metalloproteinases/inhibitors MMP-1, MMP-3, MMP-13/TIMP-1 were evaluated in h-MSCs grown on plastic or on HA-based scaffold by Real-time PCR, ELISA, and immunocytochemical techniques. Moreover, the expression of two HA receptors, CD44 and CD54, was analyzed. We found both at mRNA and protein levels that HA-based scaffold induced the expression of CXCR4, CXCL13, and MMP-3 and downmodulated the expression of CXCL12, CXCR5, MMP-13, and TIMP-1 while HA-based scaffold induced CD54 expression but not CD44. We found that these two HA receptors were directly involved in the modulation of CXCL12, CXCL13, and CXCR5. This study demonstrates a direct action of a 3-D HA-based scaffold, widely used for cartilage and bone repair, in modulating both h-MSCs inflammatory and degradative factors directly involved in the engraftment of specific cell types in a damaged area. Our data clearly demonstrate that HA in this 3-D conformation acts as a signaling molecule for h-MSCs.

To evaluate the role of CXC chemokines CXCL8 (IL8), CXCL10 (IP-10), CXCL12 (SDF-1), and CXCL13 (BCA-1) in bone remodeling, we analyzed their effects on osteoblasts (OBs) obtained from subchondral trabecular bone tissue of osteoarthritis (OA) and post-traumatic (PT) patients. The expression of CXC receptors/ligands (CXCR1/CXCL8, CXCR2/CXCL8, CXCR3/CXCL10, CXCR4/CXCL12, and CXCR5/CXCL13) was analyzed in cultured OBs by flow cytometry and immunocytochemistry. Functional assays on CXC chemokine-treated-OBs in the presence or absence of their specific inhibitors were performed to analyze cellular proliferation and the enzymatic response to chemokine activation. The expression of chemokine ligands/receptors was also confirmed in bone tissue samples by immunohistochemical analysis. Collagen type I and alkaline phosphatase mRNA expression were analyzed on CXCL12- and CXCL13-treated OBs by real-time PCR. OBs from both OA and PT patients expressed high levels of CXCR3 and CXCR5 and lower amounts of CXCR1 and CXCR4. CXCL12 and CXCL13, only in OBs from OA patients, induced a significant proliferation that was also confirmed by specific blocking experiments. Moreover, OBs from OA patients released a higher amount of CXCL13 than those of PT patients while no differences were found for CXCL12. In the remodeling area of bone tissue samples, immunohistochemical analysis confirmed that OBs expressed CXCL12/CXCR4 and CXCL13/CXCR5 both in OA and PT samples. CXCL12 and CXCL13 upregulated collagen type I mRNA expression in OBs from OA patients. These data suggest that CXCL12 and CXCL13 may directly modulate cellular proliferation and collagen type I in OA patients, so contributing to the remodeling process that occurs in the evolution of this disease., (Copyright 2005 Wiley-Liss, Inc.)

Chemokines are involved in a number of inflammatory pathologies and some of them show a pivotal role in the modulation of osteoclast development. Therefore, we evaluated the role of CXCL12 chemokine on osteoclast differentiation and function and we analyzed its expression on synovial and bone tissue biopsies from rheumatoid arthritis (RA) patients. Osteoclasts were obtained by 7 days in vitro differentiation with RANKL and M-CSF of CD11b positive cells in the presence or absence of CXCL12. The total number of osteoclast was analyzed by Tartrate-resistant acid phosphatase (TRAP)-staining and bone-resorbing activity was assessed by pit assay. MMP-9 and TIMP-1 release was evaluated by ELISA assay. CXCL12 expression on biopsies from RA patients was analyzed by immunohistochemistry. Osteoclasts obtained in the presence of CXCL12 at 10 nM concentration displayed a highly significant increase in bone-resorbing activity as measured by pit resorption assay, while the total number of mature osteoclasts was not affected. The increased resorption is associated with overexpression of MMP-9. Immunostaining for CXCL12 on synovial and bone tissue biopsies from both rheumatoid arthritis (RA) and osteoarthritis (OA) samples revealed a strong increase in the expression levels under inflammatory conditions. CXCL12 chemokine showed a clear activating role on mature osteoclast by inducing bone-resorbing activity and specific MMP-9 enzymatic release. Moreover, since bone and synovial biopsies from RA patients showed an elevated CXCL12 expression, these findings may provide useful tools for achieving a full elucidation of the complex network that regulates osteoclast function in course of inflammatory diseases.

The mechanism by which osteoblasts (OB) interact and modulate the phenotype and proliferation of T lymphocytes during inflammation is not well known. The effects of two regulatory cytokines, TNFalpha and IFNgamma, on the expression of CD54 (ICAM-1) and CD106 (VCAM-1) adhesion molecules and the CXCR3 ligands (CXCL9, CXCL10, CXCL11), were assessed in a primary culture of human OB by real-time PCR, flow cytometry, and immunohistochemistry. In addition, we functionally evaluated the recruitment and proliferation of T lymphocytes grown with resting or stimulated OB. According to the present data IFNgamma, either alone or in combination with TNFalpha, significantly up-regulates the expression of CD54 and CD106 and induces the expression and release of CXCL9, CXCL10, CXCL11 in OB. The supernatant of TNFalpha- and IFNgamma-activated OB induces the recruitment of T lymphocytes more significantly than stimulation by CXCR3 ligands. T lymphocyte proliferation is significantly enhanced by direct contact with TNFalpha- and IFNgamma-activated OB or by incubation with the supernatant of TNFalpha- and IFNgamma-activated OB. Blocking experiments with anti-CD11a, anti-CD49d, anti-CXCR3, and Bordetella pertussis toxin demonstrate that adhesion molecules and the CXCR3 chemokine receptor play a key role in the proliferation of T lymphocytes. The present study demonstrates the involvement of adhesion molecules (CD11a and CD49d) and chemokine receptor (CXCR3) in the mechanism by which OB recruit, interact, and modulate T lymphocyte proliferation under inflammatory conditions., (Copyright 2003 Wiley-Liss, Inc.)

Osteoblasts (OBs) contribute to the maintenance of bone homeostasis and their activity can be influenced by immune cells localized in bone lacunae. We investigated the expression of the chemokine receptors in isolated human OBs by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, and report a novel finding, namely, that OBs express high levels of CXC chemokine receptor 3 (CXCR3) and 5 (CXCR5). Functional assays to evaluate CXCR3 and CXCR5 demonstrated that their ligands-CXCL10 and CXCL13, respectively-significantly induce the release of beta-N-acetylhexosaminidase, an enzyme involved in endochondral ossification and bone remodeling able to degrade important extracellular matrix components. Alkaline phosphatase activity, a useful index of matrix formation was also up-regulated by CXCL10 and CXCL13. However, OB activation by these ligands does not affect OB proliferation. Both Bordetella pertussis toxin and neutralizing anti-CXCR3/anti-CXCR5 monoclonal antibodies block CXCL10 and CXCL13 induction, respectively. We also demonstrated the expression of CXCL10 and CXCL13 in human bone tissue biopsies. These results indicate that both CXCR3/CXCL10 and CXCR5/CXCL13 receptor-ligand pairs may play an important role in OB activity through the specific up-regulation of two enzymes, which are involved in the bone remodeling process. Moreover, our data suggest that OBs may play a role in the modulation of bone formation through the combined action of these two enzymes., (Copyright 2002 Wiley-Liss, Inc.)