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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.

Bone homeostasis is regulated by cells at different stages of maturation that are influenced by soluble factors. The modulatory function of two pro-inflammatory cytokines, IL-1beta and TNF-alpha, on the expression of CXCL13 chemokine was evaluated in osteoblasts (OB) and bone marrow stromal cells (BMSC) from osteoarthritis (OA) and post-traumatic (PT) patients. In basal condition, CXCL13 production by both BMSC and OB was significantly higher in OA than in PT patients. IL1beta, significantly induced CXCL13 production in differentiated OB, both from OA and PT patients, but not in BMSC from both either group. TNFalpha reduced CXCL13 production only in BMSC from OA patients. The combination of IL1beta and TNFalpha increased CXCL13 production only in OB in the same amount as for IL-1beta alone. OB from OA released a higher amount of CXCL13 compared to PT in all conditions tested. CD121a (IL1 receptor type I) was highly expressed only by OB. Moreover, in bone tissue biopsies CXCL13 was expressed by mesenchymal and mononuclear cells. This study demonstrates that cells at different stages of maturation (BMSC and OB) and derived from physiological (PT) or pathological conditions (OA) respond in different ways to inflammatory stimuli. These data may contribute to understand the basic maturation processes of bone cells in old patients.

Cartilage tissue engineering remains problematic because no systems are able to induce signals that contribute to native cartilage structure formation. Therefore, we tested the potentiality of gelatin-polyethylene glycol scaffolds containing three different concentrations of chitosan (CH; 0%, 8%, and 16%) on chondrogenic differentiation of human platelet lysate-expanded human bone marrow mesenchymal stromal cells (hBM-MSCs). Typical chondrogenic (SOX9, collagen type 2, and aggrecan), hypertrophic (collagen type 10), and fibrotic (collagen type 1) markers were evaluated at gene and protein level at Days 1, 28, and 48. We demonstrated that 16% CH scaffold had the highest percentage of relaxation with the fastest relaxation rate. In particular, 16% CH scaffold, combined with chondrogenic factor TGFβ3, was more efficient in inducing hBM-MSCs chondrogenic differentiation compared with 0% or 8% scaffolds. Collagen type 2, SOX9, and aggrecan showed the same expression in all scaffolds, whereas collagen types 10 and 1 markers were efficiently down-modulated only in 16% CH. We demonstrated that using human platelet lysate chronically during hBM-MSCs chondrogenic differentiation, the chondrogenic, hypertrophic, and fibrotic markers were significantly decreased. Our data demonstrate that only a high concentration of CH, combined with TGFβ3, creates an environment capable of guiding in vitro hBM-MSCs towards a phenotypically stable chondrogenesis.

This paper aims to characterize the wear behavior of hydrogel constructs designed for human articular cartilage replacement. To this purpose, poly (ethylene glycol) diacrylate (PEGDA) 10% w/v and gellan gum (GG) 1.5% w/v were used to reproduce the superior (SUP) cartilage layer and PEGDA 15% w/v and GG 1.5% w/v were used to reproduce the deep (DEEP) cartilage layer, with or without graphene oxide (GO). These materials (SUP and DEEP) were analyzed alone and in combination to mimic the zonal architecture of human articular cartilage. The developed constructs were tested using a four-station displacement control knee joint simulator under bovine calf serum. Roughness and micro-computer tomography (&micro, CT) measurements evidenced that the hydrogels with 10% w/v of PEGDA showed a worse behavior both in terms of roughness increase and loss of uniformly distributed density than 15% w/v of PEGDA. The simultaneous presence of GO and 15% w/v PEGDA contributed to keeping the hydrogel construct&rsquo, s characteristics. The Raman spectra of the control samples showed the presence of unreacted C=C bonds in all the hydrogels. The degree of crosslinking increased along the series SUP &lt, DEEP + SUP &lt, DEEP without GO. The Raman spectra of the tested hydrogels showed the loss of diacrylate groups in all the samples, due to the washout of unreacted PEGDA in bovine calf serum aqueous environment. The loss decreased along the series SUP &gt, DEEP + SUP &gt, DEEP, further confirming that the degree of photo-crosslinking of the starting materials plays a key role in determining their wear behavior. &mu, CT and Raman spectroscopy proved to be suitable techniques to characterize the structure and composition of hydrogels.

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin–chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin–chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin–chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%–90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin–chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin–chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.

The pathways that control mesenchymal stem cells (MSCs) differentiation are not well understood, and although some of the involved transcription factors (TFs) have been characterized, the role of others remains unclear. We used human MSCs from tibial plateau (TP) trabecular bone, iliac crest (IC) bone marrow and Wharton’s jelly (WJ) umbilical cord demonstrating a variability in their mineral matrix deposition, and in the expression levels of TFs including Runx2, Sox9, Sox5, Sox6, STAT1 and Slug, all involved in the control of osteochondroprogenitors differentiation program. Because we reasoned that the basal expression level of some TFs with crucial role in the control of MSC fate may be correlated with osteogenic potential, we considered the possibility to affect the hMSCs behaviour by using gene silencing approach without exposing cells to induction media. In this study we found that Slug-silenced cells changed in morphology, decreased in their migration ability, increased Sox9 and Sox5 and decreased Sox6 and STAT1 expression. On the contrary, the effect of Slug depletion on Runx2 was influenced by cell type. Interestingly, we demonstrated a direct in vivo regulatory action of Slug by chromatin immunoprecipitation, showing a specific recruitment of this TF in the promoter of Runx2 and Sox9 genes. As a whole, our findings have important potential implication on bone tissue engineering applications, reinforcing the concept that manipulation of specific TF expression levels may elucidate MSC biology and the molecular mechanisms, which promote osteogenic differentiation.

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.

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. J. Cell. Physiol. 226: 982–990, 2011. © 2010 Wiley-Liss, Inc.

Surface properties affect the biological properties of cells modulating the expression of different factors. Osteoblasts contribute both to new bone formation and controlling haematopoiesis through cytokines and growth factors. We analyzed the effect of bone (calcium-phosphate bone slides), cartilaginous (hyaluronan-based scaffold), and plastic substrate culture on human osteoblast proliferation, bone matrix molecule, and inflammatory factor expression. Osteoblast proliferation increased to a greater extent when the cells were grown for 14 days on plastic and bone slides, whereas hyaluronan-based scaffold (HA-scaffold) induced only a minimal increase. Collagen type I, osteonectin, alkaline phosphatase and osteocalcin were expressed on osteoblasts grown on plastic and bone slides and down-modulated at mRNA and protein level by HA-scaffold. Bone slides showed the ability to increase osteopontin mRNA expression. The expression of CXCR4 and CXCL13 was upregulated by bone slides and HA-scaffold, while CXCL12 and CXCR5 expression was down-modulated. These data suggest a substrate-dependent modulation of human osteoblast proliferation, bone matrix and inflammatory factor expression, which might help to understand the active role played by osteoblasts in bone microenvironment by coupling bone extracellular matrix, chemokines and the haematopoietic system.

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.

no abstract available

We evaluated the role of CCL20 (MIP-3α) 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. J. Cell. Physiol. 210: 798–806, 2007. © 2006 Wiley-Liss, 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.

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. J. Cell. Physiol. 226: 2675–2682, 2011. © 2011 Wiley-Liss, Inc.

CXCL12/CXCR4 chemokine/receptor axis signaling has recently been found to play an important role in the remodeling of bone tissue, but little is known about the molecular mechanisms that are involved. The present study shows that CXCL12 is present at high levels both in human mesenchymal stem cells (hMSCs) and primary osteoblasts (hOBs). When osteogenesis was induced, CXCL12 expression was strictly confined to mineralized nodules. To investigate what mechanisms contribute to the maintenance of a correct expression of CXCL12 in bone cellular context, we analyzed the relationship between CXCL12 and Slug, a transcription factor recently associated with osteoblast maturation. By gene silencing and chromatin immunoprecipitation assay, we showed that both proteins are required for the mineralization process and CXCL12 is transcriptionally and functionally regulated by Slug, which is recruited at specific sites to its gene promoter in vivo. These findings showed for the first time a positive correlation between CXCL12 signaling and Slug activity, thus corroborating the role of these two proteins in bone cellular context and suggesting a new potential target for bone tissue repair and regeneration.

Bone osteoblastic and mesenchymal stromal cells lack primarily tumoral features in multiple myeloma patients

This study aims to define the function of Slug transcription factor in human normal osteoblasts (hOBs). To date, Slug is considered exclusively a marker of malignancy in bone tissue. Here, we identified, for the first time, a role for Slug in hOBs using a knockdown approach. We demonstrated that Slug is positively correlated with osteoblast markers, including Runx2, osteopontin, osteocalcin, Collagen type 1, Wnt/beta-catenin signaling mediators, and mineral deposition. At the same time, Slug silencing potentiates the expression of Sox-9, a factor indispensable for chondrogenic development. These data, with the finding that Slug is in vivo recruited by the promoters of Runx2 and Sox-9 genes, suggest that, in hOBs, Slug may act both as positive and negative transcriptional regulator of Runx2 and Sox-9 genes, respectively. In summary, our results support the hypothesis that Slug functions as a novel regulator of osteoblast activity and may be considered a new factor required for osteoblast maturation.

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.

Osteoarthritis (OA) is a disease that limits the mobility of patients and is of considerable economical importance. Up to now, despite the increasing number of patients with OA, treatments to manage the disease remain symptomatic, designed to control pain, and improve function and quality of life limiting adverse events. With the aim to explore a new approach to treat OA patients suffering from early degenerative lesions of hyaline cartilage, we transplanted in an experimental animal model of OA a hyaluronan-based scaffold (Hyaff11) seeded with mesenchymal stem cells (MSCs) obtained from bone marrow and expanded in culture.Rabbit knee joints were bilaterally subjected to anterior cruciate ligament transection to surgically induce OA. After 8 weeks, the time necessary to the development of cartilage surface damage, animals were treated with MSCs seeded onto Hyaff-11 scaffold in the left condyle and unseeded Hyaff-11 in the controlateral knee. Untreated rabbits were used as controls. All the animals were sacrificed at 3 and 6 months after surgery. Histological, histomorphometric, and immunohistological evaluations were performed.OA changes developed in all animals subjected to anterior cruciate ligament transection. The predominant macroscopically observed OA changes were mild (lateral femoral condyle) or moderate (medial femoral condyle) ulcerations. Statistically significant differences in the quality of the regenerated tissue were found between the implants with scaffolds carrying MSCs compared to the scaffold alone or controls in particular at 6 months.From the observations, it is possible to demonstrate that Hyaff-11, a hyaluronan-based scaffold, has potential for MSC implantation and that may have application for the treatment of early OA in humans.

Human mesenchymal stem cells (hMSCs) are an alternative cell source in bioconstruct production for cartilage regeneration, and hyaluronic acid (HA) is a widely-used bioabsorbable scaffold material used for cartilage regeneration. In this work, the aims were to evaluate the mechanical competency of hMSC-seeded HA scaffolds compared with native intact human articular cartilage, and in relation to its cellular properties. Human MSCs were grown under static conditions in HA scaffolds and then tested, in stepwise, stress-relaxation indentation, 7, 14, and 21 days later. Scaffolds at days 14 and 21 showed a significant increase in mechanical measures when compared with day 7 and unseeded scaffold material, but did not achieve the same levels as human cartilage. There was consistent stiffness within the scaffold, with a decreased stiffness around the edge. In vitro culture of hMSC-seeded HA scaffolds over 3 weeks produces a white, solid tissue compared with unseeded constructs. Increased cell proliferation and collagen type II expression were also seen over this period of time. These results demonstrate the competency of the neo-formed cartilage-like tissue in relation to its mechanical and cellular properties, and further, the importance, for future clinical use, of implanting this construct after 14 days of culture.

Chemokines contribute to the maintenance of cartilage homeostasis. To evaluate the role of CXC chemokines CXCL8 (interleukin-8), CXCL10 (interferon-gamma-inducible protein-10), CXCL12 (stroma-derived factor-1) and CXCL13 (B-cell attracting chemokine-1) and their receptors, respectively CXCR1-2, CXCR3, CXCR4, and CXCR5, during chondrogenic differentiation of human mesenchymal stromal cells (h-MSCs), we used a well-defined in vitro model. Chondrogenic differentiation was analyzed on h-MSCs grown on hyaluronic acid-based biomaterial in the presence or absence of transforming growth factor-beta, and the expression and modulation of CXC chemokines and receptors were evaluated at different time points. Real-time polymerase chain reaction was performed to analyze their expression at the messenger ribonucleic acid (mRNA) level, and immunohistochemistry and enzyme-linked immunosorbent assay were used to evaluate their expression at the protein level. Human articular cartilage biopsies were used to evaluate chemokine and receptor expression in normal tissue. We found no expression of CXCR1, CXCR2, CXCR3, or CXCL10 at the mRNA level. CXCL8 mRNA was down-modulated, whereas at the protein level, we found greater release of this chemokine. CXCR4 and its ligand CXCL12 were down-modulated during chondrogenesis. By contrast, CXCR5 was up-regulated, whereas its ligand CXCL13 was lower. These data were also confirmed on human articular cartilage. These findings show that, during in vitro h-MSC chondrogenic differentiation, chemokine and receptor expression was specifically induced or repressed. This was in line with what the authors also found in normal articular cartilage, suggesting a role in differentiation and maturation of a cartilage-like structure in vitro and consequently the regulation of cartilage homeostasis.

The expression of the chemokine CC-chemokine ligand 20 (CCL20)/macrophage inflammatory protein (MIP)-3α and its receptor CC-chemokine receptor 6 (CCR6) by multiple myeloma (MM) and microenvironment cells and their potential relationship with osteoclast (OC) formation and osteolytic bone lesions in MM patients was investigated in this study. First, we found that MM cells rarely produce CCL20/MIP-3α but up-regulate its production by bone marrow (BM) osteoprogenitor cells and osteoblasts in coculture with the involvement of soluble factors as interleukin-1β and tumor necrosis factor α. MM cells also stimulate both CCL20/MIP-3α and CCR6 expression by OCs in coculture. Thereafter, we showed that CCL20/MIP-3α significantly increases both the number of multinucleated tartrate-resistant acid phosphatase–positive OCs and receptor activator of nuclear factor-κB–positive OC progenitor cells similar to CCL3/MIP-1α. Finally, we found that blocking anti-CCL20/MIP-3α and anti-CCR6 antibodies significantly inhibits MM-induced OC formation. In vitro data were further expanded in vivo analyzing a total number of 64 MM patients. Significantly higher CCL20/MIP-3α levels were detected in MM patients versus monoclonal gammopathy of uncertain significance (MGUS) subjects and in MM osteolytic patients versus nonosteolytic ones. Moreover, a significant increase of CCL20/MIP-3α–positive osteoblasts in osteolytic MM patients compared with nonosteolytic ones was observed. Interestingly, no significant difference in BM CCL20/MIP-3α expression and level was observed between MGUS and nonosteolytic MM patients. Our data indicate that CCL20/MIP-3α and its receptor CCR6 are up-regulated in the bone microenvironment by MM cells and contribute to OC formation and osteolytic bone lesions in MM patients. [Cancer Res 2008;68(16):6840–50]

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.

Phosphoinositides play an essential role in diverse cellular functions such as cell proliferation, cytoskeletal regulation, intracellular vesicle trafficking, motility, cell metabolism and death. Alteration of these pathways is common to many diseases. In this study, we show that osteoblasts from patients affected by osteoarthritis (OA) and by rheumatoid arthritis (RA) present a decreased cell proliferation and a reduced expression of the key elements of polyphosphoinositide signal transduction such as phosphatidylinositol-3-kinase (PI 3K), phospholipase C gamma1 (PLCgamma1), and protein kinase C zeta (PKCzeta) compared to the post-traumatic (PT) patients. Our results suggest that a correlation may exist between the reduced osteoblast proliferation observed in OA and RA patients and the lowered expression of PI 3K, PLCgamma1, and PKCzeta enzymes. The reduced proliferation rate of osteoblasts in response to these signal transduction effectors could counteract the evolution of arthritic disease.

Mesenchymal stromal cells (MSCs) seem to be a good alternative to chondrocytes for cartilage regeneration. To obtain new information on the sequence of cellular and molecular events during in vitro chondrogenic differentiation we analysed MSCs on a widely used hyaluronic acid biomaterial (Hyaff ® -11). Cellular differentiation was induced using two different concentrations of TGF β 1 (10 and 20 ng/ml) and the process was analysed at different time points (24 h, and 7, 14, 21 and 28 days) using techniques of light and electron microscopy, real-time PCR and immunohistochemistry. We found that without TGF β MSCs did not survive while in the presence of TGF β the cells significantly proliferated from day 7 until day 28. Light and electron microscopy showed that TGF β at 20 ng/ml better induced the formation of cartilage-like tissue. Real-time PCR showed an increased expression of collagen type II, IX and aggrecan associated to a down-regulation of collagen type I. Immunohistochemical analysis confirmed that collagen type I was down-modulated while collagen type II increased from day 14 to day 28. These data clearly showed that higher concentrations of TGF β (20 ng/ml) induce chondrogenesis of MSCs on Hyaff ® -11 scaffold better than 10 ng/ml of TGF β . This process is characterized by a sequence of cellular and molecular events that deal with the in vitro formation of a cartilage-like tissue.

The objective of this study was to define the effects of osteoarthritic (OA) milieu on good manufactured practice‐adipose‐derived mesenchymal stromal cells (GMP‐ASC) that are commonly utilized in cell therapies. Two different OA milieu: OA synovial fluid (SF) and OA‐conditioned medium (CM) from synoviocytes were used to treat GMP‐ASC both in normoxia or hypoxia. GMP‐ASC were tested for cell migration, proliferation, cytokine receptors expression (CXCR1, CXCR2, CXCR3, CXCR4, CXCR7, CCR1, CCR2, CCR3, CCR5, IL6R), and cytokines (CXCL8/IL8, CXCL10/IP10, CXCL12/SDF‐1, CCL2/MCP1, CCL3/MIP1α, CCL4/MIP1β, CCL5/RANTES, IL6) release. Healthy SF was used as controls. We demonstrated that GMP‐ASC show an increase in proliferation, migration, and modulation of CXCR1, CXCR3, CCR1, and CCR5 receptors in hypoxic condition. Moreover, GMP‐ASC migration increased 15‐fold when treated either with OA‐SF or OA‐CM compared with healthy SF both in normoxia and hypoxia. GMP‐ASC treated in both OA milieu showed an increase in CXCR3, CCR3, and IL6R and a decrease in CCR1 and CCR2 receptors. In OA‐SF, we detected higher amount of CXCL10/IP10 than in OA‐CM, while CCL2/MCP1 and CCL4/MIP1β were higher in OA‐CM compared with OA‐SF. CXCL10/IP10 was the only chemokine of the OA milieu, which was down‐modulated after treatment with GMP‐ASC. In conclusion, we demonstrated specific effects of OA milieu on both GMP‐ASC proliferation, migration, and cytokine receptor expression that were strictly dependent on the inflammatory and hypoxic environment. The use of characterized OA milieu is crucial to define the therapeutic effect of GMP‐ASC and indicates that CXCL10/IP10–CXCR3 axis is partially involved in the GMP‐ASC effect on synovial macrophages. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:336‐347, 2020, The objective of this study was to define in vitro the effects of osteoarthritic (OA) milieu and hypoxia on good manufactured practice‐adipose‐derived mesenchymal stromal cells (GMP‐ASC) that are commonly utilized in cell therapies. Hypoxia increased GMP‐ASC proliferation. OA milieu‐treated GMP‐ASC showed an increase in cellular migration, as well as in CXCR3, CCR3, and IL6R and a decrease in CCR1 and CCR2 receptors in hypoxia. CXCL10/IP10 was the only chemokine of the OA milieu down‐modulated after treatment with GMP‐ASC.