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Background In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β. Methodology/Principal Findings The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13. Conclusions/Significance Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.

PRP cryopreservation remains a controversial point. Our purpose was to investigate the effect of freezing/thawing on PRP molecule release, and its effects on the metabolism of chondrocytes and synoviocytes. PRP was prepared from 10 volunteers, and a half volume underwent one freezing/thawing cycle. IL-1β, HGF, PDGF AB/BB, TGF-β1, and VEGF were assayed 1 hour and 7 days after activation. Culture media of chondrocytes and synoviocytes were supplemented with fresh or frozen PRP, and, at 7 days, proliferation, gene expression, and secreted proteins levels were evaluated. Results showed that in the freeze-thawed PRP the immediate and delayed molecule releases were similar or slightly lower than those in fresh PRP. TGF-β1 and PDGF AB/BB concentrations were significantly reduced after freezing both at 1 hour and at 7 days, whereas HGF concentration was significantly lower in frozen PRP at 7 days. In fresh PRP IL-1βand HGF concentrations underwent a significant further increase after 7 days. Similar gene expression was found in chondrocytes cultured with both PRPs, whereas in synoviocytes HGF gene expression was higher in frozen PRP. PRP cryopreservation is a safe procedure, which sufficiently preserves PRP quality and its ability to induce proliferation and the production of ECM components in chondrocytes and synoviocytes.

In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Background: Platelet-rich plasma (PRP) has been advocated as one treatment for cartilage tissue regeneration. To date, several different platelet-rich formulations have been available, but a deep knowledge of their composition and mechanism of action in a specific clinical use is needed. The aim of this study was to investigate the effect of various PRP formulations on human chondrocytes in vitro. Methods: Blood from ten human volunteers was used to prepare three formulations: (1) PRP with a relatively low concentration of platelets and very few leukocytes (P-PRP), (2) PRP with high concentrations of both platelets and leukocytes (L-PRP), and (3) platelet-poor plasma (PPP). Selected growth factors in the formulations were measured, and the in vitro effects of various concentrations were tested by exposing chondrocytes isolated from osteoarthritic cartilage of four different men and measuring cell proliferation, matrix production, and gene expression. Results: L-PRP contained the highest levels of growth factors and cytokines. All three formulations stimulated chondrocyte proliferation throughout the culture period evaluated; the only significant difference among the formulations was on day 7, when P-PRP induced greater cell growth compared with the other two formulations. P-PRP stimulated chondrocyte anabolism, as shown by the expression of type-II collagen and aggrecan, whereas L-PRP promoted catabolic pathways involving various cytokines. However, L-PRP induced greater expression of the hyaluronic acid synthase-2 gene and greater production of hyaluronan compared with P-PRP. Conclusions: L-PRP and P-PRP induced distinct effects on human articular chondrocytes in vitro, possibly because of differences in the concentrations of platelets, leukocytes, growth factors, and other bioactive molecules. The identification of the optimal amounts and ratios of these blood components could ideally lead to a formulation more suitable for the treatment of cartilage lesions. Clinical Relevance: PRP formulations that are suitable for the treatment of degenerative joint diseases will likely contain balanced concentrations of platelets and leukocytes.

Background aims. Platelet-rich plasma (PRP), a blood derivative rich in platelets, is a relatively new technique used in tissue regeneration and engineering. The increased quantity of platelets makes this formulation of considerable value for their role in tissue healing and microbicidal activity. This activity was investigated against five of the most important strains involved in nosocomial infections (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Streptococcus faecalis) to understand the prophylactic role of pure (P)-PRP. Microbicidal proteins released from activated P-PRP platelets were also determined. Methods. The microbicidal activity of P-PRP and platelet-poor plasma (PPP) was evaluated on different concentrations of the five bacterial strains incubated for 1, 2, 4 and 18 h and plated on agar for 18e24 h. P-PRP and PPP-released microbicidal proteins were evaluated by means of multiplex beadebased immunoassays. Results. P-PRP and PPP inhibited bacterial growth for up to 2 h of incubation. The effect of P-PRP was significantly higher than that of PPP, mainly at the low seeding concentrations and/or shorter incubation times, depending on the bacterial strain. Chemokine (CC motif) ligand-3, chemokine (C-C motif) ligand-5 and chemokine (C-X-C motif) ligand-1 were the molecules mostly related to Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis inhibition. Escherichia coli and Klebsiella pneumoniae were less influenced. Conclusions. The present results show that P-PRP might supply an early protection against bacterial contaminations during surgical interventions because the inhibitory activity is already evident from the first hour of treatment, which suggests that physiological molecules supplied in loco might be important in the time frame needed for the activation of the innate immune response.

An elevation of b-galactoside a2,6-sialyltransferase(ST6Gal.I) enzyme activity and an increased a2,6-sialyla-tion of cell membranes are among the most prominentglycosylation changes associated with colon cancer; bothmodifications correlate with a worse prognosis. In ourprevious studies, we have frequently observed a discrepancybetween the ST6Gal.I level within a colon cancer sample orcell line and the respective level of reactivity with thea2,6-sialyl-specific lectin from Sambucus nigra (SNA). Inthis study, we have investigated quantitatively thebiosynthesis of the sialyl-a2,6-lactosaminyl epitope in twocolon cancer cell types expressing the ST6Gal.I cDNAunder the control of a constitutive promoter. By measuringthe amount of ST6Gal.I mRNA using competitive RT-PCR,the expression of a2,6-sialylated lactosaminic structureswith SNA and anti-CDw75 Ig, and the presence ofunsubstituted lactosaminic termini by Erythrina cristagallilectin, we reached the following conclusions: (a) a highproportion of the cell surface lactosaminic termini remainsunsubstituted, even in the presence of a very high ST6Gal.Iactivity. This proportion is strongly dependent on the celltype; (b) ST6Gal.I-transfected colon cancer cells do notexpress the CDw75 epitope; (c) the level of ST6Gal.Ienzyme activity only partially correlates with the mRNAlevel; (d) despite the control by a constitutive promoter, theST6Gal.I mRNA is not constantly expressed over time; and(e) a very large portion of the enzyme molecules is secretedin the extracellular milieu. These results indicate that post-transcriptional and post-translational mechanisms play apivotal role in the control of a2,6-sialylation in colon cancercells.Keywords: colon cancer; glycosyltransferases; glycosyla-tion; a2,6-sialyltransferase; CDw75.The biosynthesis of the oligosaccharide moieties ofglycoconjugates is a complex process and the fine regulationof this process is only partially understood. Although it isgenerally thought that the biosynthesis of a given oligo-saccharide structure is regulated mainly by the expression ofthe cognate glycosyltransferase, the enzyme–productrelationship might in some cases correlate very poorly[1,2], suggesting that other factors play a relevant role.b-Galactoside a2,6-sialyltransferase (ST6Gal.I) is theonly enzyme able to catalyze the a2,6-sialylation oflactosamine (Galb1,4GlcNAc) [3], a structure commonlyfound in the antennae of N-linked chains but present also inO-linked chains and in glycolipids (reviewed in [4]). One ofthe most characteristic glycosylation changes in coloncancer is represented by an elevation of ST6Gal.I enzymeactivityincancertissues,incomparisonwiththesurrounding normal mucosa [5]. Clinical studies indicatethat ST6Gal.I enzyme activity is further increased inmetastatic tissues [6] and that a high level of ST6Gal.I inprimary cancer is associated with a poor prognosis [7]. Also,the level of a2,6-sialylation of membrane glycoconjugates,as determined by the binding of NeuAca2,6Gal-specificlectins from Sambucus nigra (SNA) and Tricosanthesjaponica, is increased in colon cancer [8–10] and a strongreactivity is associated with a worse prognosis [11]. Thus, itis important to clarify the relationship between these twoparameters for potential clinical use. In recent studies, wehave observed that the level of ST6Gal.I enzyme activity ina given colon cancer sample only partially correlates withthe level of SNA reactivity, suggesting the existence of othermechanisms of regulation [12,13]. Moreover, while theincrease of ST6Gal.I enzyme activity in colon cancer is ageneral phenomenon (the exceptions constituting ,10% ofall cases), some studies reported an increase of ST6Gal.ImRNA only in a subset of cases [1,14]. Together, these datasuggest that multiple factors control the expression of theoligosaccharide product. Many of these factors might be cellspecific, for example, the stability of the mRNA, theefficiency of the translational machinery, the stability andthe post-translational processing of enzyme proteins, theexpression of other glycosyltransferases involved in thebiosynthesis of backbone oligosaccharide structures.We have undertaken this study to investigate quantitat-ively the sequence of biochemical events, from mRNAexpression to the final oligosaccharide product, in a modelsystem designed to minimize the contribution of these

Human natural killer (NK) cells constitutively express the beta- and gamma-chains of the interleukin 2 (IL-2)/IL-15 receptor, and both IL-2 and IL-15 are able to activate NK cell proliferation and cytotoxicity. When IL-2-primed human NK cells are exposed to sensitive targets (i.e. K562) they undergo apoptosis mediated by the beta(2)-integrin CD18. Here, we demonstrate that: (i) endothelial cells, similar to K562 tumour target cells, induce apoptosis of IL-2-primed NK cells; (ii) endothelial- and K562 cell-induced apoptosis is significantly lower in IL-15 than in IL-2-stimulated NK cells; (iii) a critical role in the apoptosis of IL-2-primed NK cells is played by the alpha-chain of the IL-2 receptor. Our data show for the first time that IL-2-activated NK cells can die by apoptosis upon contact with the vascular endothelium, which is a necessary step for their extravasation, with a direct pathophysiological relevance on the strategy of adoptive immunotherapy of cancer. On the other hand, IL-15, although generating a similar level of activation of NK cells, largely prevents their apoptotic fate. Therefore, IL-15 produced early in the immune response, when T cells are not yet activated, generates lymphokine-activated killer cells that are efficient killers relatively protected from apoptosis. Once activated, T cells produce IL-2 that overcomes the effect of IL-15 on NK cells, paving the way for their death by apoptosis.

Cytotoxicity assays provide an in vitro evaluation of the lytic activity of NK and T cells against tumors or transformed cells. However, none of these methods allow the recovery of cells or supernatants after the assay. We standardized a microcytotoxicity test using calcein-acetoxymethyl (calcein-AM) dye that requires very small quantities of cells while maintaining the same sensitivity as the traditional51Cr assay. The assay is applicable to resting as well as activated human effector cells and uses different targets such as human cell lines that are adherent or growing in suspension and resistant or sensitive. The most important feature of the method is the possibility of recovering cells and supernatants for additional analyses such as phenotyping and evaluation of soluble factors.

Osteoarthritis and rheumatoid arthritis are characterized by focal loss of cartilage due to an up-regulation of catabolic pathways, induced mainly by pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumour necrosis factor alpha (TNFalpha). Since reactive oxygen species are also involved in this extracellular-matrix-degrading activity, we aimed to compare the chondrocyte oxidative status responsible for cartilage damage occurring in primarily degenerative (osteoarthritis) and inflammatory (rheumatoid arthritis) joint diseases. Human articular chondrocytes were isolated from patients with osteoarthritis or rheumatoid arthritis, or from multi-organ donors, and stimulated with IL-1beta and/or TNFalpha. We evaluated the oxidative stress related to reactive nitrogen and oxygen intermediates, measuring NO(-)(2) as a stable end-product of nitric oxide generation and superoxide dismutase as an antioxidant enzyme induced by radical oxygen species. We found that cells from patients with osteoarthritis produced higher levels of NO(-)(2) than those from patients with rheumatoid arthritis. In addition, IL-1beta was more potent than TNFalpha in inducing nitric oxide in both arthritides, and TNFalpha alone was almost ineffective in cells from rheumatoid arthritis patients. We also observed that the intracellular content of copper/zinc superoxide dismutase (Cu/ZnSOD) was always lower in rheumatoid arthritis chondrocytes than in those from multi-organ donors, whereas no differences were found in intracellular manganese SOD (MnSOD) or in supernatant Cu/ZnSOD and MnSOD levels. Moreover, intracellular MnSOD was up-regulated by cytokines in osteoarthritis chondrocytes. In conclusion, our results suggest that nitric oxide may play a major role in altering chondrocyte functions in osteoarthritis, whereas the harmful effects of radical oxygen species are more evident in chondrocytes from patients with rheumatoid arthritis, due to an oxidant/antioxidant imbalance.

Physical activity has been reported to affect endocrine function in elderly men.To establish an association between regular moderate physical activity and endogenous anabolic hormone levels in healthy aging men.Twenty four middle-aged (57.4+/-4.7 years) and 24 elderly (68.3+/-2.6 years) physically active men who in the past 10 years had been regularly bicycling during leisure time were compared with 24 middle-aged (57.9+/-4.0 years) and 24 elderly (67.2+/-1.7 years) sedentary men. Groups did not differ for body composition.Serum dehydroepiandrosterone sulfate (DHEAS), insulin-like growth factor-I (IGF-1), free testosterone (FT), and thyroid hormone levels were assessed.In general, elderly men had lower IGF-1 (P0.001), DHEAS (P=0.013), and triodothyronine levels (P0.001) than their middle-aged counterparts. Independently of age, however, physically active men had on average higher IGF-1 (P=0.031), DHEAS (P=0.001), and triodothyronine serum levels (P0.001) than sedentary men. FT and thyroid stimulating hormone (TSH) serum concentrations did not differ across age groups, but physically active men had lower TSH values than sedentary men (P=0.021).Our results suggest that, in aging men, regular moderate physical activity is associated with higher levels of IGF-1 and DHEAS levels and with thyroid function alterations.

The function of chemokines in promoting and modulating leukocyte migration is essential for a prompt and efficacious inflammatory response and in host defence against infections. In order to investigate whether this important aspect of immunological response is influenced by ageing, we evaluated the basal levels as well as the ability of peripheral blood mononuclear cells from young and healthy elderly subjects to produce chemokines (IL-8, MCP-1, MIP-Ialpha, RANTES) in response to stimulation with anti-CD3 monoclonal antibody and lipopolysaccharide (LPS), a gram negative bacterial endotoxin. Our main findings are a spontaneous chemokine production; a 20% decrease of proliferative response to anti-CD3 monoclonal antibody accompanied by an age related increase of MIP-Ialpha and RANTES production and by a general increase of all chemokine production compared to unstimulated conditions; a proliferative defect of monocytes to LPS challenge associated with an increase of chemokine production compared to basal conditions with a progressive age-related increase of MIP-lalpha. In conclusion, this study suggests that chemokines could have a compensatory role in balancing the impaired mechanisms involved in 'specific' immune response during ageing. The successful activation of this strategy could contribute to the good performance of immune system so maintaining healthy status in elderly.

SUMMARY The determination of serum levels of antibodies against hepatitis B virus surface antigen (anti-HBs) after hepatitis B vaccination is currently the only simple test available to predict the decay of protection and to plan the administration of booster doses. A total of 3085 vaccine recipients of plasma-derived and recombinant vaccine have been followed for 10 years to determine the kinetics of anti-HBs production and to construct a mathematical model which could efficiently predict the anti-HBs level decline. The anti-HBs peak level was reached 68 days after the last dose of recombinant vaccine and 138 days after the last dose of plasma-derived vaccines. The age of vaccinees negatively influenced the anti-HBs levels and also the time necessary to reach the anti-HBs peak. A bilogarithmic mathematical model (log10 level, log10 time) of anti-HBs decay has been constructed on a sample of recombinant vaccine recipients and subsequently validated on different samples of recombinant or plasma-derived vaccine recipients. Age, gender, type of vaccine (recombinant or plasma-derived), number of vaccine doses (three or four) did not influence the mathematical model of antibody decay. The program can be downloaded at the site: http://www2.stat.unibo.it/palareti/vaccine.htm. Introducing an anti-HBs determination obtained after the peak, the program calculates a prediction of individual anti-HBs decline and allows planning of an efficient booster policy.

SUMMARY Polymyalgia rheumatica (PMR) is a common disorder of the elderly: the pathogenesis of the syndrome is still debated, though active synovitis of the shoulder has recently been confirmed. To investigate the pathogenesis of this synovitis we evaluated cell adhesion molecule (CAM) expression in shoulder synovial tissue from patients with PMR, correlated synovial expression with the serum levels of soluble forms, and assessed the changes associated with corticosteroid treatment. Arthroscopic synovial biopsies were obtained from 12 untreated and seven corticosteroid (CS)-treated cases. CAM expression was evaluated by MoAb staining on frozen sections and computerized image analysis. Soluble CAM were quantified by ELISA. Endothelial cells expressed intercellular adhesion molecule-1 (ICAM-l), E- and P-selectins. Infiltrating cells were ICAM-1 and β1-integrin-positive, while L-selectin expression was limited to intravascular leucocytes. Synovial lining cells strongly expressed vascular cell adhesion molecule-1 (VCAM-1), and less intensely ICAM-1. Only the soluble form of ICAM-1 (sICAM-1) was elevated in untreated patients. CS treatment was associated with a decrease in ICAM-1, VCAM-1 and E- and P-selectin expression. sICAM-1 levels were in the normal range in treated patients. VLA-5 and 6 expression was widely distributed among cell types, and was not CS-sensitive. Active shoulder synovitis is associated with different CAM expression in PMR. ICAM-l expression is widely distributed and correlates with elevated levels of the soluble form; it is significantly lower in CS-treated asymptomatic cases.

The extent of processing of N-linked oligosaccharides and the sialylation of the target cell membranes has been positively correlated with resistance to lysis mediated by NK cells, but a conclusive evidence has never been reached. Colon cancer tissues express an increased activity of beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1, alpha 2,6ST), which catalyzed the addition of sialic acid in alpha 2,6-linkage to Gal beta 1,4GlcNAc (N-acetyllactosamine) sequences of glycoprotein N-linked chains. The resulting increased level of membrane alpha 2,6-sialylation appears to be related with a more invasive behavior of cancer cells. This phenomenon may depend on a decreased sensitivity of colon cancer cells to NK cells. To obtain conclusive evidence on the role played by sialylation of N-linked chains in determining the target cell susceptibility to NK-mediated lysis, human colon cancer cell lines not expressing sialyltransferases acting on N-linked chains were transfected with a rat alpha 2,6ST cDNA. Stable transfectants expressed different levels of alpha 2,6ST activity, were reactive with the Sambucus nigra lectin, specific for alpha 2,6-linked sialic acid, and compared with control transfectants, showed a remarkable decrease in the number of unsubstituted Gal beta 1,4GlcNAc terminal sequences. The NK susceptibility of these clones was found to be identical to that of control transfectants, either when unstimulated- or IL-2-stimulated lymphocytes were used as effectors. Neuraminidase treatment of target cells does not result in significant changes to NK susceptibility. Our data demonstrate that sialic acid alpha 2,6-linked to N-linked chains of target cell glycoproteins does not play a major role in recognition of the target by human NK cells.

Un numero sempre crescente di evidenze sottolinea l’importante contributo dell’angiogenesi nell’ambito dei meccanismi che inducono le modificazioni articolari caratteristiche dell’osteoartrosi (OA). Nei pazienti con OA, alcune molecole correlate al processo angiogenetico, quali vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1) e fibroblast growth factor-2 (FGF-2) sono espresse a livello articolare e possono avere un ruolo non solo in relazione all’angiogenesi, ma anche all’omeostasi cartilaginea. Questo studio si propone di valutare i livelli circolanti di VEGF, VCAM-1 e FGF-2 in pazienti con OA della mano affetti da forma erosiva (OA-E) e non-erosiva (OA-NE) e di determinare le eventuali associazioni con il danno articolare valutato tramite analisi radiologica convenzionale. Sono stati analizzati, con metodo ELISA, i sieri di 146 pazienti con OA-E, di 148 pazienti con OA-NE e di 148 soggetti di controllo (CN). I livelli circolanti di sVCAM-1 nei pazienti con OA-E sono risultati più elevati rispetto a quelli evidenziati nei CN; non sono state dimostrate differenze tra i pazienti con OAE e i pazienti con OA-NE. L’analisi di VEGF e FGF-2 non ha evidenziato differenze significative tra pazienti con OA e CN, inoltre non sono emerse correlazioni tra i livelli circolanti di VEGF, sVCAM-1 e FGF-2 e parametri radiologici e clinici. I dati ottenuti, dimostrando un aumento dei livelli circolanti di sVCAM-1 nei pazienti con forma erosiva di OA della mano, caratterizzata da una peggiore decorso e maggiore aggressività, aggiungono nuove informazioni circa la possibilità di identificare sVCAM-1 come biomarcatore di severità di malattia.

BACKGROUND AIMS: An extensive debate about the clinical benefits of autologous platelet concentrates used as a treatment option for patients with orthopedic injuries is ongoing. The aim of this study was to determine whether different compositions of platelet concentrates may affect the osteogenic differentiation of bone marrow stromal cells (BMSC). METHODS: Pure platelet-rich plasma (P-PRP) and leukocyte-PRP (L-PRP) were characterized for platelet and leukocyte content. As an indicative marker of the delivery of growth factors (GFs), the release of basic fibroblast growth factor (bFGF) from platelet gel (PG) was measured at 1, 18, 48 and 72 h and at 7 d. The ability of different PGs to induce proliferation and differentiation of BMSC was evaluated by using bioactivity assays. RESULTS: The platelet recovery was significantly higher in L-PRP, either fresh or frozen. PGs derived from L-PRP and P-PRP showed significant differences in terms of bFGF release and biological activity. bFGF release was faster both in fresh and frozen L-PRP preparations. Moreover, L-PRP samples were able to induce a significantly higher proliferation of BMSC compared with P-PRP or PPP samples. Even though all PG preparations allowed the deposition of mineral nodules in BMSC cultures, the mineralization activity correlated significantly with bFGF levels. CONCLUSIONS: The biological activity of platelet concentrates differs according to preparation technique, which affects platelet and leukocyte content and GF availability. Because GF levels are not always optimal in subjects with defective bone healing, composition and bioactivity of PRP should be analyzed to test the reliability and potential effectiveness of the regenerative treatment. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved

Background: Fresh osteochondral allografts represent a treatment option for early ankle posttraumatic arthritis. Transplanted cartilage survivorship, integration, and colonization by recipient cells have not been fully investigated. The aim of this study was to evaluate the ability of recipient cells to colonize the allograft cartilage and to assess allograft cell phenotype. Methods: Seventeen ankle allograft samples were studied. Retrieved allograft cartilage DNA from fifteen cases was compared with recipient and donor constitutional DNA by genotyping. In addition, gene expression was evaluated on six allograft cartilage samples by means of real-time reverse transcription-polymerase chain reaction. Histology and immunohistochemistry were performed to support molecular observations. Results: Of fifteen genotyped allografts, ten completely matched to the host, three matched to the donor, and two showed a mixed profile. Gene expression analysis showed that grafted cartilage expressed cartilage-specific markers. Conclusions: The rare persistence of donor cells and the prevailing presence of host DNA in retrieved ankle allografts suggest the ingrowth of recipient cells into the allograft cartilage, presumably migrating from the subchondral bone, in accordance with morphological findings. The expression of chondrogenic markers in some of the samples argues for the acquisition of a chondrocyte-like phenotype by these cells. Clinical Relevance: To our knowledge, this is the first report describing the colonization of ankle allograft cartilage by host cells showing the acquisition of a chondrocyte-like phenotype.

DNA ploidy was investigated in 61 specimens obtained from 25 patients with squamous carcinoma of the oral and maxillofacial region. Biopsy specimens of normal tissue surrounding the tumor were also obtained in six patients. Single-cell suspensions for flow cytometric analysis were prepared. The DNA ploidy and histogram were calculated and compared with the histologic grade, presence of lymph node metastases, and course of the disease. The ploidy of the main stemline was peridiploid in 17 carcinomas, hyperdiploid in three, and aneuploid in five. Histologic grade but not nodal involvement was associated with the ploidy of the main stemline. Of 15 multisampled carcinomas 13 showed constant DNA ploidy and histogram classification. In the other two major changes in DNA ploidy (from peridiploid to hyperdiploid in the first and from peridiploid to aneuploid in the second) were found. Survival information was available for 24 patients. Ploidy values higher or lower than 2.5 c were strongly predictive of both overall (p < 0.001) and relapse-free survival (p < 0.001). The lymph node status proved a powerful prognostic indicator (p = 0.014) but was not related to the relapse-free time of survival. Multiparametric evaluation of survival revealed an independent role of both DNA ploidy and nodal involvement in the prognosis of squamous carcinoma of the oral and maxillofacial region.

Objective: To investigate the immunologic features of synovitis in patients with polymyalgia rheumatica (PMR) and to assess the modifications induced by corticosteroid. Methods. Arthroscopic biopsies of shoulder synovium were obtained from 12 patients with untreated PMR and from 7 patients with PMR that had been treated. Immunohistochemistry was performed on frozen sections utilizing a panel of monoclonal antibodies and computerized image analysis. Results. Synovitis was present in 10 of 12 (83%) untreated patients and in only 2 of 7 (29%) treated patients. The synovitis was characterized by vascular proliferation and leukocyte infiltration. Infiltrating cells consisted predominantly of macrophages and T Lymphocytes. Almost all T lymphocytes were CD45RO positive. A few neutrophils, but no B cells, natural killer cells, or γ/δ T cells were found. Intense expression of HLA class II antigens (DR moreso than DP moreso than DQ) was found in the lining layer cells as well as in macrophages and lymphocytes. DR, but not DP or DQ, was expressed by the endothelium of a few vessels. Class II antigen expression correlated with the number of macrophages and lymphocytes. Macrophage infiltration of arteriole walls was observed in 1 untreated patient without giant cell arteritis (GCA). In untreated patients, there was a positive correlation between the percentage of infiltrating T cells and the duration of disease. Steroid therapy was associated with a significant reduction in the number of blood vessels and of HLA class II expression. One treated patient who no longer had symptoms of PMR still had active synovitis: a relapse occurred 4 months after the biopsy. Conclusion. Our findings support the hypothesis that synovitis is a major cause of the musculoskeletal symptoms of PMR. There are immunologic similarities with the vascular inflammation observed in GCA. Corticosteroids act on both the vascular and cellular components of synovitis.

Peripheral lymphoid tissues contain a fibroblastic cell type referred to as stromal cells or reticulum cells which interact with lymphocytes as part of the lymphoid microenvironment. After isolation from human tonsils and expansion in vitro we analyzed the surface phenotype, extracellular matrix components, cytoskeletal products, cytokine production, binding and functional interaction with B lymphocytes of in vitro cultured stromal cells (HTSC) both in resting condition and after activation with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our results show that HTSC do not express specific myeloid, lymphoid, endothelial or epithelial markers. HTSC express CD54 (ICAM-1), CD49a (VLA-1), CD49b (VLA-2), CD49c (VLA-3), CD49e (VLA-5), CD49f (VLA-6), CD29, CD51, CD44 and produce vinculin, beta-tubulin, alpha-actin, vimentin, fibronectin, laminin and collagen types I, III and IV. Activation of HTSC up-regulated CD54 (ICAM-1) and induced HLA-DR and CD106 (VCAM-1). HTSC constitutively produce interleukin (IL)-6 which is enhanced upon activation with TNF-alpha. IL-8 and granulocyte/macrophage colony-stimulating factor are detected only in the supernatants of activated HTSC. Reverse transcriptase polymerase chain reaction analysis revealed that HTSC display mRNA for IL-1 alpha, leukemia inhibitory factor and IL-7. The adhesion of tonsillar B lymphocytes to activated HTSC is mediated by CD11a/CD18 and CD54. Furthermore, HTSC can induce maximal proliferation of IL-2-activated B lymphocytes cocultured in direct cell-cell contact with HTSC. These results clearly distinguish in vitro cultured HTSC from common fibroblasts and other non-lymphoid elements present in the lymphoid parenchyma, such as follicular dendritic cells, and show that HTSC actively participate in the lymphoid microenvironment. In vitro cultures of HTSC could therefore be a useful model system for detailed analysis of the interactions between stromal cells and lymphocytes under physiological and pathological conditions.

T lymphocytes play a key role in the regulation of bone homeostasis and bone healing. The inflammatory response at the site of bone injury is essential to the initiation of the bone repair program; however, an uncontrolled exposure to inflammatory environment has a negative effect on tissue regeneration - indeed, activated T cells were shown to inhibit osteogenic differentiation on human mesenchymal stromal cells (MSCs). Whether resting T cells can induce osteogenic differentiation of MSCs and what role specific T cells subset play in this process is still elusive. In this study, we sought to analyse the osteogenic gene expression profile of whole T cells, CD4 and CD8 T cells isolated from healthy donors and investigated whether secreted factors from each group modulate osteogenic differentiation of human MSCs. Gene expression profiling identified a pool of 51 genes involved at various stages in bone growth which are expressed above detectable levels in CD4 and CD8 T cells. Most genes of this pool were expressed at higher levels in the CD4 subset. In vitro mineralization assays revealed that conditioned medium from CD4 T cells, but not from CD8 cells, significantly increased mineralization in osteogenic cultures of human MSCs; furthermore, mRNA expression of Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) in MSCs was significantly upregulated in the presence of CD4-conditioned medium but not with that obtained from CD8. The results show a differential role for CD4 and CD8 T cells in supporting bone formation and identify an osteogenic gene signature of each subset.

In this study, we investigated the efficacy of the transplantation of autologous articular chondrocytes seeded onto a hyaluronan-based scaffold, known as Hyaff(®)-11, on the treatment of early cartilage lesions in a rabbit osteoarthritis (OA) model. The hypothesis of the study was that this treatment could enhance cartilage repair after OA induction. OA was surgically induced by Anterior Cruciate Ligament Transection (ACLT) in thirty rabbits. Animals were divided into three groups, according to treatment: group 1: ACLT; group 2: Chondrocytes+Hyaff-11; and group 3: Hyaff-11 alone. The animals were euthanized, respectively, at 3 and 6 months after the treatment. Histomorphometrical analyses were performed by means of fibrillation index, cartilage thickness, and subchondral bone thickness evaluations. Histological appearance was scored according to Modified Kraus' Mankin and Osteoarthritis Research Society International (OARSI) scores. Immunohistochemical analyses were carried out for type I and II collagens, MMP-1, and MMP-3. A comparison between groups and follow-up for each outcome was performed with the general linear model with Sidak correction. Histomorphometrical evaluations at 3 and 6 months demonstrated that OA lesions became significantly worse followed by Hyaff-11 treatment, whereas Chondrocytes+Hyaff-11 treatment had the best overall subjective grade. Overall raw histological scores demonstrated a significant improvement with chondro-hyaluronic acid (HA) treatment at 3 months compared with HA in cartilage repair processes. Immunohistochemical analyses displayed a strong positivity for type II collagen in the Chondrocytes+Hyaff-11group at 3 months compared with the HA group. No staining was observed in MMP-3 expression in this group at any experimental point. The use of Chondrocytes+Hyaff-11 for the treatment of early OA lesions produced, already at 3 months, a repair tissue showing better macroscopic, histological, and immunohistochemical results than those observed after Hyaff-11 treatment. Thus, the transplantation of Chondrocytes+Hyaff-11 would seem to be advocated in the very early stage of the OA disease when a precocious action is mandatory.

The main purpose of this article was to describe the morphology of mesenchymal stem cells (MSCs) differentiated in vitro towards osteogenic and chondrogenic lineages and to focus on the ultrastructural features associated with these processes. Human mononuclear cells (hMNC) were isolated, expanded, and analyzed for the expression of specific cell surface markers to demonstrate their stem cell characteristics. Human mononuclear cells were differentiated in vitro in an osteogenic and in a chondrogenic sense for 7, 14, 21, and 28 days. Subsequently, they were processed using electron microscopic analysis (FEISEM). Alizarin red and alcian blue staining were carried out to demonstrate the deposition of mineral salts and proteoglycans in the extracellular matrix. Undifferentiated MSCs showed a cell surface covered by filopodia and ondulopodia. During differentiation, the MSCs changed their shape from a round to a fibroblastic-like shape. At the end of the differentiation, several filaments with a parallel orientation in the osteogenic samples as well as a network organization in the chondrogenic samples were detected in the extracellular spaces. This study demonstrated that there are morphological features associated with the undifferentiated and differentiated states of the MSCs, which could be utilized as new parameters for identifying and classifying these cells. © 2011 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.

PURPOSE: Early OA primarily affects articular cartilage and involves the entire joint, including the subchondral bone, synovial membrane, menisci and periarticular structures. The aim of this review is to highlight the molecular basis and histopathological features of early OA. METHODS: Selective review of literature. RESULTS: Risk factors for developing early OA include, but are not limited to, a genetic predisposition, mechanical factors such as axial malalignment, and aging. In early OA, the articular cartilage surface is progressively becoming discontinuous, showing fibrillation and vertical fissures that extend not deeper than into the mid-zone of the articular cartilage, reflective of OARSI grades 1.0-3.0. Early changes in the subchondral bone comprise a progressive increase in subchondral plate and subarticular spongiosa thickness. Early OA affects not only the articular cartilage and the subchondral bone but also other structures of the joint, such as the menisci, the synovial membrane, the joint capsule, ligaments, muscles and the infrapatellar fat pad. Genetic markers or marker combinations may become useful in the future to identify early OA and patients at risk. CONCLUSION: The high socioeconomic impact of OA suggests that a better insight into the mechanisms of early OA may be a key to develop more targeted reconstructive therapies at this first stage of the disease

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.

The present study describes, for the first time, the removal of the nuclear factor of activated T cells cytoplasmic 1 (NFATc1) by a decoy approach in human primary osteo - blasts (hOBs). hOBs with different NFATc1 expression levels were used. The functionality of endogenous NFAT proteins in our experimental model was analyzed by monitoring the tran - scriptional activity on a luciferase reporter construct driven by three copies of an NFAT response element (pNFAT-TA-luc). Cell treatment with the decoy against NFATc1 resulted in a significant increase in the expression of osteoblastic markers, including ERα and ColXV. On the contrary, the expression of Runx2, which is known to not be transcriptionally regulated by NFATc1, was not altered, indicating the specificity of the decoy effect. To our knowledge, this is the first time that tran- scription factor decoy has been successful in hOBs to allow the investigation of the role of NFATc1 in an experimental model that, compared to the use of cell lines, more closely resembles an in vivo model. In addition, by using chro- matin immunoprecipitation we found that in vivo NFATc1 is recruited on the ColXV gene promoter. The specific role of NFATc1 in osteoblast differentiation is not well understood, however, our findings reinforce the action of NFATc1 in the transcriptional program of osteoblasts, also supporting the therapeutic potential for the proper manipulation of NFATc1- mediated events in different bone disorders. At the same time, our data add important information on the regulation of the expression of ColXV, which only recently has been proposed as an osteoblastic marker.

Background How to assess the impact of research is of growing interest to funders, policy makers and researchers mainly to understand the value of investments and to increase accountability. Broadly speaking the term "research impact" refers to the contribution of research activities to achieve desired societal outcomes. The aim of this overview is to identify the most common approaches to research impact assessment, categories of impact and their respective indicators. Methods We systematically searched the relevant literature (PubMed, The Cochrane Library (1990-2009)) and funding agency websites. We included systematic reviews, theoretical and methodological papers, and empirical case-studies on how to evaluate research impact. We qualitatively summarised the included reports, as well the conceptual frameworks. Results We identified twenty-two reports belonging to four systematic reviews and 14 primary studies. These publications reported several theoretical frameworks and methodological approaches (bibliometrics, econometrics, ad hoc case studies). The "payback model" emerged as the most frequently used. Five broad categories of impact were identified: a) advancing knowledge, b) capacity building, c) informing decision-making, d) health benefits, e) broad socio-economic benefits. For each proposed category of impact we summarized a set of indicators whose pros and cons are presented and briefly discussed. Conclusions This overview is a comprehensive, yet descriptive, contribution to summarize the conceptual framework and taxonomy of an heterogeneous and evolving area of research. A shared and comprehensive conceptual framework does not seem to be available yet and its single components (epidemiologic, economic, and social) are often valued differently in different models.

In this study, we investigated genetic damage acquisition during in vitro culture of human osteoarthritic (OA) chondrocytes to evaluate their safety for use in regenerative medicine clinical applications. In particular, we have addressed the impact of long-term in vitro culture on simple sequence repeat stability, to evaluate the involvement of the mismatch repair system (MMR) in the accumulation of genetic damage. MMR, the main post-replicative correction pathway, has a fundamental role in maintaining genomic stability and can be monitored by assessing microsatellite instability (MSI). MMR activity has been reported to decrease with age not only in vivo, but also in vitro in relationship to culture passages. OA chondrocytes from seven donors were cultured corresponding to 13-29 population doublings. Aliquots of the cells were collected and analyzed for MSI at five DNA loci (CD4, VWA, FES, TPOX, and P53) and for MMR gene expression at each subculture. Genetic stability was confirmed throughout the culture period. MMR genes demonstrated a strong coordination at the transcriptional level among the different components; expression levels were very low, in accordance with the observed genetic stability. The reduced expression of MMR genes might underline no need for increasing DNA repair control in the culture conditions tested, in which no genetic damage was evidenced. These data argue for the safety of chondrocytes for cellular therapies and are encouraging for the potential use of in vitro expanded OA chondrocytes, supporting the extension of autologous cell therapy procedures to degenerative articular diseases.

OBJECTIVE: To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. METHODS: MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and beta-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. RESULTS: Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, beta-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13-remodeled or -degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. CONCLUSION: MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.

Impaired lymphocyte and granulocyte function in the aged may, in part, reflect intrinsic aged-related biochemical alterations. In this study we compared the ribonucleotide contents of lymphocytes and granulocytes from young and old subjects evaluated by means of an HPLC-anion exchange method. We found that in general both populations from old subjects present higher levels of the various nucleotides, in particular: ATP, UDP, CTP, UDP-glucose in granulocytes, AMP, CTP, UDP-N-acetylglucosamine, UDP-glucose in lymphocytes. These data suggest that these molecules accumulate in aged subjects because of altered biochemical pathways. The increased pool of UDP-sugars, in particular, could be due to a depressed activity of some glycosyltransferases which therefore fail to glycosylate some plasma membrane cell proteins, thus accounting for their functional impairment.

The recognition of effector cell populations that are able to actively form conjugates with target cells is of major importance in studies of lymphocyte cytotoxicity. A number of methodologies have been described to identify the conjugates and count them, but there have been few studies of the birding capability of the different subsets of effector cells involved in the conjugation phenomenon. Here we describe a methodology that permits the study of two surface markers on lymphocytes conjugated to K562 target cells. In particular, the expression of low density CD8 (CD8 dim ) has been studied on both CD3 + and CD16 + lymphocytes bound to K562 target cells. Previously described methodologies, either optical microscopy or flow cytometry, were not able to identify the effector population by mAb double staining, especially in the case of antigens expressed at low density. The flow cytometric methodology described here permits the measurement of the binding activity of small lymphocyte subsets such as the CD3 + 8 dim+ population. However, the method could be used to study the binding activity of any effector population defined by mAb double staining.

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.

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.

The opportunity to apply autologous chondrocyte transplantation in repairing cartilage lesions in osteoarthritis (OA) is of great interest. To this end, chondrocytes from cartilage of these patients and from healthy donors were used to evaluate the expression of some extracellular matrix molecules once these cells were grown onto a hyaluronan-based scaffold already used in clinical practice. Constructs were analyzed by immunohistochemical and real-time PCR analyses. Chondrocytes from control and patients with OA cartilages expressed the same extracellular matrix molecules even if at different amount. These differences, which were appreciable both at protein and molecular levels, were not evident once the cells were grown onto Hyaff-11 scaffold. In this experimental culture condition, the cells derived from control and patients with OA showed a significant increase of collagen type II, Sox-9, and aggrecan and a decrease of collagen type I compared with chondrocytes grown in monolayer. On the other hand, MMPs were downregulated in both the cell types evaluated by the specific action of TIMP-1 which was highly expressed at molecular and protein levels in the two groups. The growth of chondrocytes onto Hyaff-11 membrane seems to erase the differences between the cells derived from normal and OA cartilages. The cells seem to benefit of the "hyaluronan" presence which is able to create an ideal environment for the expression of cartilage genes even in absence of specific growth factors. This is of particular relevance hypothesizing the use of tissue engineering therapeutical approach also in patients with OA.

Chondrocyte apoptosis is known to contribute to articular cartilage damage in osteoarthritis (OA) and is correlated to a number of cartilage disorders. Micromass cultures represent a convenient means for studying chondrocyte biology, and, in particular, their death. In this study, we present the first ultrastructural analysis on chondrocyte death experimentally induced by different agents, all known to be powerful apoptotic triggers. Chondrocytes were obtained from subjects undergoing joint arthroplasty. At the end of the maturation, they were treated as follows: 10 or 30 μM etoposide for 24h, UV-B for 30 min followed by 4h recovery, 200 or 500 nM staurosporine for 24h, hyperthermia for 1h at 43°C followed by 4h recovery. They were processed for TEM and immunofluorescence. Control chondrocyte morphology appears similar to that of human articular cartilage. Proteoglycans and collagen fibers are present in the intercellular space, indicating a good extracellular matrix (ECM) production. Etoposide, when effective, induces necrosis. UV-B treated cells show chromatin condensation and pore clustering, typical of apoptotic nuclei. Hyperthermia seems to induce apoptosis in the presence of abundant ECM, giving, differently, a general necrosis when ECM is scarse. Staurosporine has no effect at 200 nM concentration, but frequently, at 500 nM, chromatin condensation appears. Chromatin clumping appears different from that of classical apoptotic models: Roach et al. proposed the term “chondroptosis” to indicate this type of cell death. Cells appear shrunk and the nucleus contains condensed chromatin, not marginated into large solid masses, but in small patches, mainly at nuclear periphery. Chondrocytes seem so to undergo apoptosis in a peculiar typical manner.

Pro-inflammatory cytokine response and NK activity are controlled by the availability of zinc ion, whose intra-cellular transport is regulated by metallothioneins. In order to closely examine the importance of circulating zinc in the modulation of immune response during ageing, in the balance of Th2/Th1 equilibrium and finally in the reversibility of systemic low grade inflammation, we evaluated the changes occurring in plasma IL-6 and MCP-1 concentrations and NK lytic activity in a healthy low grade inflamed elderly population, following zinc-aspartate supplementation. In addition, we aimed to highlight the potential interaction among circulating zinc increments, changes in immunological parameters and +647 MT1a and −174 IL-6 polymorphic alleles. Thirty-nine healthy individuals (60–83 years) from the ZINCAGE cohort (previously typed for +647 MT1a and −174 IL-6 polymorphisms) were supplied with zinc-aspartate. Blood samples collected before and after supplementation underwent basal laboratory determinations (circulating zinc, albumin and C-reactive protein) and immunological studies (plasma IL-6 and MCP-1 and NK lytic activity). Zinc supplementation in subjects with low or borderline-normal circulating zinc increased the concentration of this ion and modulated plasmatic IL-6 and MCP-1 as well as NK lytic activity. An interactive effect of polymorphic alleles of MT1a and IL-6 genes on zinc, IL-6, MCP-1 and NK activity was evidenced following supplementation, indicating the genetic background as one of the determinants for identifying groups of subjects that can take advantage of therapeutic intervention.

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 treatment of large (6 cm) segmental bone defects, especially in areas associated with soft tissue damages and exposure, is challenging. Massive bone allografts are widely used in reconstructive surgery to replace missing bone parts such as critical size defects, nevertheless, early vascular invasion is a critical factor in bone allograft incorporation and in the consequent success of the surgery. Unfortunately, it has been proven that only slow and incomplete bone integration is possible by using frozen allografts in the treatment of large bone defects. An allogenic frozen bone graft was engineered with bone marrow mononuclear cells (BMMCs) to repair a large ulna defect in a male patient. When infection and sinus developed at the graft site 4 months after implantation, the partially reabsorbed graft was removed and send to the lab for examination. The histological and immunohistochemical analysis performed on a graft removed from the central zone of the defect demonstrated areas of neo-vascularisation, indicating that a remodelling process was actively occurring even in an area not usually repopulated with the conventional techniques. For these reasons, we may hypothesise that the use of BMMCs has a role in the re-building of large segmental defects.

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]

The DNA mismatch repair system, the main postreplicative correction pathway in eukaryotic cells, has been shown to be involved in the acquisition of genetic damage during the aging of normal somatic cells, including those of the immune system. Previously, we showed that some but not all human T cell clones (TCC) in an in vitro culture aging model develop microsatellite instability (MSI), which is associated with altered expression of mismatch repair genes. Here, we analyzed levels of mismatch repair proteins as well as the corresponding mRNAs and related this to the development of microsatellite instability in TCC. Msh2, Msh3, Msh6, Pms1, and Pms2 protein expression was quantified by Western blotting. We found that clones not manifesting microsatellite instability in this in vitro model of T cell replicative aging, induced by persistent antigenic stimulation, maintain normal transcriptional control and coordination among the mismatch repair system genes, while clones which do manifest MSI display a general deregulation of gene expression, which is likely to contribute to its occurrence.

We compared the secretion of vascular endothelial growth factor (VEGF) by chondrocytes isolated from cartilage of patients with osteoarthritis (OA) and maintained in monolayer or in three-dimensional culture. Chondrocytes, immediately after isolation, or after one passage in monolayer culture (subculture), were seeded in monolayer or pelleted in micromasses. Cells cultured differently were immunoassayed for the secretion of VEGF in basal conditions or after stimulation with IL-17, TNF-alpha or B IL-17+TNF-alpha. Chondrocytes cultured in micromasses secreted lower levels of VEGF in comparison with those in monolayer culture. In micromass cultures TNF-alpha was more stimulating than IL-17 and their combined effect was additional. On the basis of alkaline phosphatase/cathespin B activities, primary cultures in micromass, characterized by low VEGF secretion, represented the best condition for maximal cell differentiation. Our results suggest that the culture of chondrocytes in micromasses represents a good condition to study the physiology of these cells to a maximum degree when micromasses are obtained with freshly isolated chondrocytes.

An intense vascularization of primary tumor mass is associated with a fatal outcome in various types of invasive solid tumors. Interleukin 17 (IL-17), a CD4+ T-cell-derived cytokine, stimulates some tumor cells to secrete angiogenic factors, among which venous endothelial growth factor (VEGF). We assessed whether the expression of IL-17 receptor (IL-17R) represents a marker for the metastasizing ability of osteosarcoma (OS), a very malignant bone tumor. We immunoassayed the amount of VEGF secreted by three OS cell lines expressing IL-17R in differing amounts: HOS, MG63 and U-2 OS, and their sensitivity to IL-17 stimulation to secrete VEGF. U-2 OS, which best expresses IL-17R, secreted the highest amounts of VEGF and was the most sensitive to IL-17, whereas MG63 expressed the lowest level of IL-17R, secreted the lowest amount of VEGF and was not sensitive to IL-17. IL-17R expression correlated with VEGF secretion and IL-17 sensitivity. U-2 OS expressed the most dedifferentiated phenotype, which is associated with tumor malignancy. These results suggest that IL-17R in OS might represent a marker of tumor metastasis potential.

Chemokines play an important role in orchestrating leukocyte recruitment and activation during inflammation. Given the ubiquity of chemokines involved in inflammatory tissue destruction, it is not surprising that they contribute to numerous human pathologies. Epidemiological studies have suggested that chronic low-grade inflammation is related to several diseases of ageing with an inflammatory pathogenesis (such as atherosclerosis, type 2 diabetes, osteoarthritis and Alzheimer’s disease) and to increased mortality risk. In this chapter, we will briefly review the properties of chemokines and their receptors and highlight the roles of these chemoattractants in the above selected diseases of ageing.