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The use of megaprostheses or custom-made devices is an established treatment for or- thopedic malignancies, but their indication has been expanded to some non-neoplastic conditions such as complex cases of re-revision surgery or failed osteosynthesis. A con- secutive series of 5 patients were treated with megaprostheses as a solution for complex cases of re-revision surgery around the knee joint. Each patient was assessed clinically and radiographically at 1, 3, 6, and 12 months after surgery. One patient died for intestinal infarc- tion after surgery. The 4 remaining patients resumed gait and knee function. We did not observe infections or mobilizations of the implants, and the only complication we described was patellar dislocation of the extensor mechanism when the implant with proximal tibia resection was used. Complex re-revision surgery after TKA represents a major challenge for the orthopedic surgeon due to poor bone stock and the presence of prosthetic revision components. The implant of megaprostheses or custom-made devices can play a crucial role in these rare but complex cases of non-oncological orthopedic surgery ensuring early functional recovery.

Introduction An increasing number of patients is annually undergoing total hip arthroplasty (THA), and a significant proportion of these patients are elderly and consequently at a higher risk of complications because of age, osteoporosis, and medical comorbidities. Periprosthetic femoral fractures (PFFs) are one of the worst complications of THA associated with high rates of unfavorable prognosis. Besides, in the last decade, a new independent disease entity called “atypical femoral fracture” (AFF) has been identified and defined by the American Society for Bone and Mineral Research (ASBMR) task force. Some PFFs present clinical history and radiographic aspect consistent with an AFF, meeting the ASBMR criteria for the diagnosis of AFF except that PFFs by themselves are an exclusion criterion for AFF. However, there is an increasing number of published studies suggesting that periprosthetic atypical femoral fractures (PAFFs) exist and should not be excluded by definition. Significance Nowadays, although there is an increasing interest in PAFFs, there are still very few studies published on the topic and a lack of consensus regarding their treatment. This narrative literature review aims to introduce this new emerging topic to a wider readership describing the characteristics of PAFFs and the state-of-the-art in their management. Conclusions Many authors agree that PAFFs should be considered as a subgroup of PFFs that have atypical characteristics; they also show a significant correlation with prolonged bisphosphonate use. A correct diagnosis is paramount for proper treatment of the disease that requires both surgical and medical actions to be taken.

Background Some periprosthetic femoral fractures (PFFs) present history and radiographic aspect consistent with an atypical femoral fracture (AFF), fulfilling the criteria for AFF except that PFFs by themselves are excluded from the diagnosis of AFFs. The aim of this study is to evaluate in a single institution series of PFFs if any of them could be considered a periprosthetic atypical femoral fracture (PAFF), and their prevalence. Methods Surgical records were searched for PFFs around a primary hip stem from January 2013 to December 2019. Cases were classified according to Vancouver classification. Demographic and medical history was extracted. Fisher’s exact test was used for statistical analysis. Results One hundred fifteen PFFs were identified, 59 of them were type B1 and 16 were type C. Radiographs and medical records were available for all patients. Twenty-four patients (32%) have been treated with bisphosphonates (BPs) for longer than 4 years. Four patients presented a fracture with characteristics of PAFF. When enlarged to all PFFs of the series, no other PAFF was found: prevalence of PAFFs was 5.3% for type B1 and C cases and 3.5% for all surgically treated PFFs. Statistical significative difference between PAFFs and PFFs was found for prolonged BP assumption and for the level of fracture clear of the stem. Conclusion Fracture with characteristics of AFFs can also happen over a prosthetic stem, configuring themselves as PAFFs, and they are related to prolonged BP use. As a correct diagnosis is mandatory for proper treatment, a revision of criteria for AFFs should be considered, accepting that PAFFs exist.

The first step in skeleton development is the condensation of mesenchymal precursors followed by any of two different types of ossification, depending on the type of bone segment: in intramembranous ossification, the bone is deposed directly in the mesenchymal anlagen, whereas in endochondral ossification, the bone is deposed onto a template of cartilage that is subsequently substituted by bone. Polyamines and polyamine-related enzymes have been implicated in bone development as global regulators of the transcriptional and translational activity of stem cells and pivotal transcription factors. Therefore, it is tempting to investigate their use as a tool to improve regenerative medicine strategies in orthopedics. Growing evidence in vitro suggests a role for polyamines in enhancing differentiation in both adult stem cells and differentiated chondrocytes. Adipose-derived stem cells have recently proved to be a convenient alternative to bone marrow stromal cells, due to their easy accessibility and the high frequency of stem cell precursors per volume unit. State-of-the-art "prolotherapy" approaches for skeleton regeneration include the use of adipose-derived stem cells and platelet concentrates, such as platelet-rich plasma (PRP). Besides several growth factors, PRP also contains polyamines in the micromolar range, which may also exert an anti-apoptotic effect, thus helping to explain the efficacy of PRP in enhancing osteogenesis in vitro and in vivo. On the other hand, spermidine and spermine are both able to enhance hypertrophy and terminal differentiation of chondrocytes and therefore appear to be inducers of endochondral ossification. Finally, the peculiar activity of spermidine as an inducer of autophagy suggests the possibility of exploiting its use to enhance this cytoprotective mechanism to counteract the degenerative changes underlying either the aging or degenerative diseases that affect bone or cartilage.

The choice of graft type in the anterior cruciate ligament (ACL) reconstruction remains a subject of controversy. The aim of this study was to assess the outcomes in ACL reconstructions performed using a four-strand hamstring tendon graft (4SHG) or a LARS ligament comparing the effectiveness of the two grafts at a medium follow-up of 8 years. This retrospective, single-centre, single surgeon study evaluated the clinical, functional and radiographic outcomes in 50 patients who underwent ACL reconstruction (25 4SHG and 25 LARS). Patients who underwent surgery after more than 6 months from injury and showed radiographically visible degenerative changes at time of surgery were excluded from the study. None of the patients underwent re-surgery in the same knee. The range of motion of the operated knee, compared to the contralateral, was good for both groups. The anterior drawer test resulted negative in 21 patients (84%) in the LARS group and eight patients (32%) in the 4SHG group (P = 0.039). The Lachman test was negative in 19 patients (76%) in the LARS group and in 11 patients (44%) in the 4SHG group (P = 0.045). Although other results of ACL reconstruction measured by Lysholm scores, IKDC evaluation, Tegner scores and radiographic images showed using a LARS graft tended to be superior to using a 4SHG, there were no statistically significant differences calculated. Our results suggest that 4 years after ACL reconstruction using a LARS ligament or 4SHG dramatically improves the function outcome, while the patients in the LARS group displayed a higher knee stability than those in the 4SHG group.

Bioethanol production from non-food lignocellulosic material is one of the most important topics in the field of biofuel production. We report on experiments with a treatment for the production of fermentable sugars from the fibrous parts of alfalfa (Medicago sativa L.), a widely used forage plant. The process is divided into several consecutive steps: ball milling, microwave assisted pretreatment in aqueous medium and enzymatic hydrolysis with a mixture of cellulase and β-glucosidase leading to the final production of fermentable sugars. This process was followed by a detailed HPLC analysis for the detection of sugars, oligomers and by-products, such as furfural, hydroxymethylfurfural and acids. Scanning electron microscopy was used for a visual characterization of the material throughout all these steps. Several plant structures were identified and their successive changes during the different treatment steps were monitored and followed.

The use of short interfering RNA (siRNA) in combination with stem cells and biocompatible scaffolds is a promising strategy in regenerative medicine. Our experimental strategy was to explore the possibility of forcing or guiding the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) by knocking down a negative regulator of chondrogenesis, Slug transcription factor (TF), thus altering cell behavior. We found that TGFβ-driven chondrogenic differentiation of hMSCs cultured onto a hyaluronan-based scaffold, HYAFF(®)-11, was strengthened after cell exposure to siRNA against Slug. Slug silencing was effective in promoting the expression of chondrogenic markers, including Col2A1, aggrecan, Sox9, LEF1, and TRPS1. In addition, we confirmed that HYAFF-11 is a good scaffold candidate for hMSC use in tissue engineering applications, and showed that it is effective in sustaining TGFβ3 treatment associated with a specific gene silencing. Interestingly, preliminary results from the experimental model described here suggested that, even in the absence of differentiation supplements, Slug silencing showed a pro-chondrogenic effect, highlighting both its potential use as an alternative to TGFβ treatment, and the critical role of the Slug TF in determining the fate of hMSCs.

Hydrogen sulfide (H2S), which recently emerged as a potent regulator of tissues and organs, is broadly produced in mammalian cells but whether it can regulate bone cell function is still elusive. The main objective of this study was to establish the role of H2S in the regulation of human osteoclast differentiation and function. Sodium hydrosulfide (NaHS), a common H2S-donor, was administered in vitro to CD11b+ human monocytes, the pool of circulating osteoclasts precursors which are critically involved in osteoclast development and function in bone. NaHS dose-dependently decreased human osteoclast differentiation at concentrations which did not induce toxicity. The inhibition of human osteoclast differentiation was associated with a down-regulation in RANKL-dependent intracellular ROS levels in human pre-osteoclasts cells. Furthermore, NaHS up-regulated NRF2 protein expression, its nuclear translocation, and the transcription of the two key downstream antioxidant genes Peroxiredoxin-1 and NAD(P)H dehydrogenase quinone 1, suggesting that NRF2 activation may inhibit human osteoclast differentiation by activating a sustained antioxidant response in osteoclast progenitors; furthermore, NRF2 activators Sulforaphane and Tert-butylhydroquinone inhibited in vitro human osteoclast differentiation. Moreover, silencing NRF2 in human pre-osteoclasts totally abolished NaHS-mediated inhibition of osteoclastogenesis, suggesting that NRF2 is essential to the inhibitory function of NaHS in osteoclast development. Finally, we found that NaHS also downregulated the RANKL/OPG mRNA ratio in human mesenchymal stem cells, the key osteoclast-supporting cells. Our results suggest that NaHS shows a potential therapeutical role in erosive diseases of bone by regulating both direct and indirect mechanisms controlling the differentiation of circulating osteoclasts precursors.

Our work aimed at evaluating the role of adipose stem cells (ASC) on chondrocytes from osteoarthritic (OA) patients and identifying the mediators involved. We used primary chondrocytes, ASCs from different sources and bone marrow mesenchymal stromal cells (MSC) from OA donors. ASCs or MSCs were co-cultured with chondrocytes in a minimal medium and using cell culture inserts. Under these conditions, ASCs did not affect the proliferation of chondrocytes but significantly decreased camptothecin-induced apoptosis. Both MSCs and ASCs from different sources allowed chondrocytes in the cocultures maintaining a stable expression of markers specific for a mature phenotype, while expression of hypertrophic and fibrotic markers was decreased. A number of factors known to regulate the chondrocyte phenotype (IL-1β, IL-1RA, TNF-α) and matrix remodeling (TIMP-1 and -2, MMP-1 and -9, TSP-1) were not affected. However, a significant decrease of TGF-β1 secretion by chondrocytes and induction of HGF secretion by ASCs was observed. Addition of a neutralizing anti-HGF antibody reversed the anti-fibrotic effect of ASCs whereas hypertrophic markers were not modulated. In summary, ASCs are an interesting source of stem cells for efficiently reducing hypertrophy and dedifferentiation of chondrocytes, at least partly via the secretion of HGF. This supports the interest of using these cells in therapies for osteo-articular diseases.

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. Copyright © 2013 John Wiley & Sons, Ltd.

Objective To examine the effect of different sources of Good Manufacturing Practice clinical grade adipose-derived mesenchymal stem cells (AD-MSCs) on inflammatory factors in osteoarthritic (OA) chondrocytes and synoviocytes. Methods AD-MSCs from infrapatellar Hoffa fat, subcutaneous (SC) hip fat, and SC abdominal fat were cocultured in Transwells with chondrocytes or synoviocytes. Inflammatory factors (interleukin-1β [IL-1β], tumor necrosis factor α, IL-6, CXCL1/growth-related oncogene α, CXCL8/IL-8, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α, and CCL5/RANTES) were evaluated by quantitative reverse transcription–polymerase chain reaction or multiplex bead–based immunoassay. The role of different immunomodulators was analyzed. Results All the inflammatory factors analyzed were down-modulated at the messenger RNA or protein level independently by all 3 AD-MSC sources or by allogeneic AD-MSCs used in coculture with chondrocytes or synoviocytes. Inflammatory factor down-modulation was observed only when AD-MSCs were cocultured with chondrocytes or synoviocytes that produced high levels of inflammatory factors, but no effect was observed in cells that produced low levels of those factors, thus highlighting a dependence of the AD-MSC effect on existing inflammation. The immunomodulators IL-10, IL-1 receptor antagonist, fibroblast growth factor 2, indoleamine 2,3-dioxygenase 1, and galectin 1 were not involved in AD-MSC effects, whereas the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway exerted a role in the mechanism of antiinflammatory AD-MSC action. Conclusion The antiinflammatory effects of AD-MSCs are probably not dependent on AD-MSC adipose tissue sources and donors but rather on the inflammatory status of OA chondrocytes and synoviocytes. AD-MSCs seem to be able to sense and respond to the local environment. Even though a combination of different molecules may be involved in AD-MSC effects, the COX-2/PGE2 pathway may play a role, suggesting that AD-MSCs may be useful for therapies in osteoarticular diseases.

Human mesenchymal stem cells (hMSCs) are pluripotent adult stem cells capable of being differentiated into osteoblasts, adipocytes, and chondrocytes. The osteogenic differentiation of hMSCs is regulated either by systemic hormones or by local growth factors able to induce specific intracellular signal pathways that modify the expression and activity of several transcription factors. Runt-related transcription factor 2 (Runx2) and Wnt signaling-related molecules are the major factors critically involved in the osteogenic differentiation process by hMSCs, and SRY-related high-mobility-group (HMG) box transcription factor 9 (SOX9) is involved in the chondrogenic one. hMSCs have generated a great interest in the field of regenerative medicine, particularly in bone regeneration. In this paper, we focused our attention on the molecular mechanisms involved in osteogenic and chondrogenic differentiation of hMSC, and the potential clinical use of hMSCs in osteoarticular pediatric disease characterized by fracture nonunion and pseudarthrosis.

Bone marrow is one of the best characterized stem cell microenvironment that contains Mesenchymal Stem Cells (MSCs). MSCs have been indicated as a new option for regenerative medicine because of their ability to differentiate into bone, cartilage and adipose tissues. However, in vitro-cultivation of MSCs could be associated with some shortcomings such as the possibility of the de-differentiation or reprogramming of the cells and the increase of the risk of infection and contaminations. To overcome these problems, a new approach is represented by the use of Bone Marrow Concentrate (BMC). This enables the implant of a cell population surrounded by its microenvironment preventing all the complications related to the in vitro-culture. Moreover, the cells within the bone marrow niche are able to regulate stem cell behavior through direct physical contact and by secreting paracrine factors. The aim of this study was to investigate the phenotype of cells within BMC and their ability to differentiate into chondrogenic lineage once seeded onto a hyaluronan-based scaffold (Hyaff-11) already used in clinic. The chondrogenic potential of BMC has been evaluated by means of morphological, histological, immunohistochemical and molecular analyses. The data obtained with the current study demonstrated that cells within BMC grown onto HYAFF-11 are able to differentiate into chondrogenic sense by the expression and production of specific extracellular molecules. These findings support the use of BMC in clinic for the repair of cartilage lesions allowing its transplantation in a “One Step” procedure. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

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.

Objective To investigate the modulation of systemic levels of soluble interleukin-6 receptor (sIL-6R) and soluble gp130 (sgp130) in untreated and treated polymyalgia rheumatica (PMR) patients during a followup period of at least 24 months in order to evaluate the relationship of these molecules with clinical outcome and their feasibility to provide a prognostic tool in clinical practice. Methods We analyzed sIL-6R and sgp130 serum levels in 93 PMR patients, and 46 age-matched normal controls, at disease onset and at 1, 3, 6, 12, and 24 months of followup during corticosteroid therapy by enzyme-linked immunosorbent assay. Results No difference in sIL-6R and sgp130 levels was observed between PMR patients and normal controls at disease onset or during followup. A significant correlation was found between the number of relapses and sIL-6R concentrations at baseline and after 1, 3, and 12 months of therapy. No correlation was found between sgp130 levels and the number of relapses. Cox multivariate analysis indicated that the best model for predicting relapses was identified by sIL-6R levels and the hemoglobin value at baseline. We found that high sIL-6R levels combined with low hemoglobin values resulted in a 10.1-fold increased risk of relapse. Conclusion Our data support the identification of a potential prognostic marker of PMR outcome that might have important implications in clinical practice. Because targeting sIL-6R with blocking antibodies has proven useful in other rheumatic disorders, our results could suggest the opportunity to evaluate sIL-6R–blocking treatment in patients with PMR and elevated levels of sIL-6R at disease onset.

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

Objective Osteoarthritic (OA) chondrocytes behave in an intrinsically deregulated manner, characterized by chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKα and IKKβ are essential kinases that activate NF-κB transcription factors, which in turn regulate cell differentiation and inflammation. This study was undertaken to investigate the differential roles of each IKK in chondrocyte differentiation and hypertrophy. Methods Expression of IKKα or IKKβ was ablated in primary human chondrocytes by retro-transduction of specific short-hairpin RNAs. Micromass cultures designed to reproduce chondrogenesis with progression to the terminal hypertrophic stage were established, and anabolism and remodeling of the extracellular matrix (ECM) were investigated in the micromasses using biochemical, immunohistochemical, and ultrastructural techniques. Cellular parameters of hypertrophy (i.e., proliferation, viability, and size) were also analyzed. Results The processes of ECM remodeling and mineralization, both characteristic of terminally differentiated hypertrophic cells, were defective following the loss of IKKα or IKKβ. Silencing of IKKβ markedly enhanced accumulation of glycosaminoglycan in conjunction with increased SOX9 expression. Ablation of IKKα dramatically enhanced type II collagen deposition independent of SOX9 protein levels but in association with suppressed levels of runt-related transcription factor 2. Moreover, IKKα-deficient cells retained the phenotype of cells in a pre–hypertrophic-like state, as evidenced by the smaller size and faster proliferation of these cells prior to micromass seeding, along with the enhanced viability of their differentiated micromasses. Conclusion IKKα and IKKβ exert differential roles in ECM remodeling and endochondral ossification, which are events characteristic of hypertrophic chondrocytes and also complicating factors often found in OA. Because the effects of IKKα were more profound and pleotrophic in nature, our observations suggest that exacerbated IKKα activity may be responsible, at least in part, for the characteristic abnormal phenotypes of OA chondrocytes.

Background: The non-canonical NF-kappa B activating kinase IKK alpha, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase), has been reported to modulate pro- or anti-inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKK alpha as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy. Methodology/Principal Findings: IKK alpha expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKK alpha(f/f):CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKK alpha in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKK alpha ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKK alpha-deficient chondrocytes. IKK alpha deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of. Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKK alpha-deficient chondrocytes was rescued by a kinase-dead IKK alpha protein mutant. Conclusions/Significance: IKK alpha acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKK alpha positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. T eta nu sigma, our findings are also of potential relevance for defining the onset and/or progression of OA disease.

The molecular mechanisms underlying spermine osteo-inductive activity on human adipose-derived stem cells (ASCs) grown in 3-dimensional (3D) cultures were investigated. Spermine belongs to the polyamine family, naturally occurring, positively charged polycations that are able to control several cellular processes. Spermine was used at a concentration close to that found in platelet-rich plasma (PRP), an autologous blood product containing growth and differentiation factors, which has recently become popular in in vitro and in vivo bone healing and engineering. Adipose tissue was surgically obtained from the hypodermis of patients undergoing hip arthroplasty. Patient age negatively affected both ASC yield and ASC ability to form 3D constructs. ASC 3D cultures were seeded in either non differentiating or chondrogenic conditions, with or without the addition of 5 mu M spermine to evaluate its osteogenic potential across 1, 2, and 3 weeks of maturation. Osteogenic medium was used as a reference. The effects of the addition of spermine on molecular markers of osteogenesis, at both gene and protein level, and mineralization were evaluated. The effects of spermine were temporally defined and responsible for the progression from the early to the mature osteoblast differentiation phases. Spermine initially promoted gene and protein expression of Runx-2, an early marker of the osteoblast lineage; then, it increased beta-catenin expression and activation, which led to the induction of Osterix gene expression, the mature osteoblast commitment factor. The finding that spermine induces ASC to differentiate toward mature osteoblasts supports the use of these easily accessible mesenchymal stem cells coupled with PRP for orthopedic applications.

In clinical orthopedics suitable materials that induce and restore biological functions together with the right mechanical properties are particularly needed for the regeneration of osteochondral lesions. For this purpose, the ideal scaffold should possess the right properties with respect to degradation, cell binding, cellular uptake, non-immunogenicity, mechanical strength, and flexibility. In addition, it should be easy to handle and serve as a template for chondrocyte and bone cells guiding both cartilage and bone formation. The aim of the present study was to estimate the chondrogenic and osteogenic capability of bone marrow concentrated derived cells seeded onto a novel nano-composite biomimetic material. These properties have been evaluated by means of histological, immunohistochemical and electron microscopy analyses. The data obtained demonstrated that freshly harvested cells obtained from bone marrow were able, once seeded onto the biomaterial, to differentiate either down the chondrogenic and osteogenic pathways as evaluated by the expression and production of specific matrix molecules. These findings support the use, for the repair of osteochondral lesions, of this new nano-composite biomimetic material together with bone marrow derived cells in a "one step" transplantation procedure.

Purpose To analyse the modifications induced by two different platelet-rich plasma (PRP) preparations on osteoarthritis (OA) synoviocytes, by documenting changes in gene expression of factors involved in joint physiopathology. Methods OA synoviocytes were cultured for 7 days in medium with different concentrations of either P-PRP (a pure platelet concentrate without leucocytes but with a limited number of platelets), L-PRP (a higher platelet concentrate containing leucocytes) or platelet-poor plasma (PPP). Gene expression of interleukin (IL)-1beta, IL-6, IL-8/CXCL8, tumour necrosis factor alpha, IL-10, IL-4, IL-13, metalloproteinase-13, tissue inhibitor of metalloproteinase (TIMP)-1, (TIMP)-3, (TIMP)-4, vascular endothelial growth factor, transforming growth factor beta1, fibroblast growth factor (FGF)-2, hepatocyte growth factor (HGF), hyaluronic acid (HA) synthases (HAS)-1, (HAS)-2, and (HAS)-3 was analysed by RT-PCR. HA production was determined in culture supernatants by ELISA. Results IL-1β, IL-8 and FGF-2 were significantly induced by L-PRP compared to both P-PRP and PPP; HGF was down-modulated by L-PRP versus both P-PRP and PPP, and an inverse dose–response influence was shown for all preparations. Expression level of TIMP-4 was lower in the presence of L-PRP compared with P-PRP. HA production and HAS gene expression did not seem to be modulated by PRP. Conclusions L-PRP is able to sustain the up-regulation of proinflammatory factors, (IL-1beta, IL-8 and FGF-2), together with a down-modulation of HGF and TIMP-4 expression, two factors that have been recognized as anti-catabolic mediators in cartilage, thus supporting the need to further optimize the PRP preparations to be applied in clinical practice.

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.

CXCR2 ligands contribute to chondrocyte hypertrophy and apoptosis, important determinants in cartilage pathophysiology. We unraveled the kinetics of signaling, biochemical, transcriptional, and morphological events triggered by GROα in human osteoarthritic chondrocytes kept in three-dimensional culture. p38 MAPK activation was assessed with a highly sensitive ELISA. Effector caspase activation was evaluated by cleavage of a fluorogenic substrate. Gene expression of key markers of hypertrophy (MMP-13, Runx-2) and matrix synthesis (aggrecan), and of cathepsin B isoform CB(-2,3) was evaluated by real time PCR. Occurrence of the morphological markers of apoptosis was investigated by transmission electron microscopy (TEM). GROα led to p38 MAPK activation in passaged chondrocytes cultured in micromass but not as a high-density monolayer. This caused the downstream triggering of chondrocyte hypertrophy (MMP-13 and Runx-2 upregulation, and calcium deposition) and apoptosis/anoikis following concurrence of matrix degrading activity, and inhibition of matrix synthesis which also involved the induction of CB(-2,3). These phenomena proved to be dependent on the co-receptor role of sulfated glycosaminoglycans (sGAG) and the activation of p38 MAPK, since they were abrogated either by preincubation with soluble chondroitin-4 sulfate or p38 MAPK inhibitors. The co-receptor role of sGAG was further demonstrated by colocalization experiments of these molecules with GROα in the stimulated micromasses. These findings suggest that extracellular matrix exerts a regulatory role in chondrocytes differentiation, and that meaningful investigation of the effects of chemokines on chondrocyte biology requires culture conditions respectful of both the differentiated status of the chondrocytes and of their three-dimensional interaction with the extracellular matrix. J. Cell. Physiol. 210: 417–427, 2007. © 2006 Wiley-Liss, Inc.

Chondrogenesis is a complex process characterized by a sequence of different steps that start with the condensation of the cells, followed by the expression of specific components, such as collagens and proteoglycans. We evaluated in vitro chondrogenic differentiation of C3H10T1/2 murine mesenchymal cells and compared them with human mesenchymal stromal cells (h-MSCs) in a hyaluronic acid scaffold. We analyzed (from day 0 to day 28) cellular morphology, proliferation, and chondrogenic/osteogenic gene expression at different time points. Our data demonstrate that, during chondrogenic differentiation, murine cells proliferate both in the absence and presence of TGF beta, while h-MSCs require the presence of this activating factor. Murine cells, even if viable, differentiate on hyaluronan scaffold, maintain a fibroblastic morphology, and form a capsule outside the scaffold. At the mRNA level, murine cells showed a decrease in collagen type 1 combined with a significant increase in collagen type H (from day 0), and aggrecan (on day 28), as found for h-MSCs. Immunohistochemical data confirmed that chondrogenic differentiation of murine cells, induced by TGF beta, occurred only in some restricted areas inside the scaffold that were positive to collagen type II, but did not show a cartilage-like tissue structure, as we had found using h-MSCs. These data demonstrate that C3H10T1/2 murine cells, widely used as a chondrogenic model, show a different sequence of chondrogenic events in hyaluronic acid scaffold, compared with primary h-MSCs.

Chondrocyte apoptosis can be an important contributor to cartilage degeneration, thereby making it a potential therapeutic target in articular diseases. To search for new approaches to limit chondrocytic cell death, we investigated the requirement of polyamines for apoptosis favored by tumor necrosis factor-alpha (TNF), using specific polyamine biosynthesis inhibitors in human chondrocytes. The combined treatment of C-28/I2 chondrocytes with TNF and cycloheximide (CHX) resulted in a prompt effector caspase activation and internucleosomal DNA fragmentation. Pre-treatment of chondrocytes with alpha-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, markedly reduced putrescine and spermidine content as well as the caspase-3 activation and DNA fragmentation induced by TNF and CHX. DFMO treatment also inhibited the increase in effector caspase activity provoked by TNF plus MG132, a proteasome inhibitor. DFMO decreased caspase-8 activity and procaspase-8 content, an apical caspase essential for TNF-induced apoptosis. Although DFMO increased the amount of active, phosphorylated Akt, inhibitors of the Akt pathway failed to restore the TNF-induced increase in caspase activity blunted by DFMO. DFMO also reduced the increase in caspase activity induced by staurosporine, but in this case Akt inhibition prevented the DFMO effect. Pre-treatment with CGP 48664, an S-adenosylmethionine decarboxylase (SAMDC) inhibitor markedly reduced spermidine and spermine levels, and provoked effects similar to those caused by DFMO. Finally DFMO was effective even in primary osteoarthritis (OA) chondrocyte cultures. These results suggest that the intracellular depletion of polyamines in chondrocytes can inhibit both the death receptor pathway by reducing the level of procaspase-8, and the apoptotic mitochondrial pathway by activating Akt.

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

Background: There are only a few studies concerning the cellular, biochemical, and genetic processes that occur during the remodeling of graft tissue after autologous chondrocyte transplantation. The purpose of the present study was to quantify the expression of genes encoding extracellular matrix proteins and regulatory factors that are essential for cell differentiation in cartilage biopsy specimens from patients who had this treatment two years previously. Methods: Two cartilage biopsy specimens from each of four patients who had been treated with autologous chondrocyte transplantation and from two multiorgan donors were used. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to evaluate the expression of types I, II, and X collagen; aggrecan; cathepsin B; and early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for matrix proteins and regulatory proteins was carried out on paraffin-embedded sections. Results: Type-I collagen mRNA was expressed in all of the samples evaluated. Type-II collagen was present in autologous chondrocyte transplantation samples but at lower levels than in the controls. Type-X collagen messenger was undetectable. Aggrecan mRNA was present in all of the samples at lower levels than in the controls, while cathepsin-B messenger levels were higher and Egr-1 and Sox-9 mRNAs were expressed at lower levels. The immunohistochemical analysis showed slight positivity for type-I collagen in all of the sections. Type-II collagen was found in all of the samples with positivity confined inside the cells, while the controls displayed a positivity that was diffuse in the extracellular matrix. Cathepsin B was slightly positive in all of the samples, while the controls were negative. Egr-1 protein was particularly evident in the areas negative for type-II collagen. Sox-9 was positive in all samples, with evident localization in the superficial and middle layers. Conclusions: In biopsy specimens from autologous chondrocyte transplantation tissue at two years, there is evidence of the formation of new tissue, which displays varying degrees of organization with some fibrous and fibrocartilaginous features. Long-term follow-up investigations are needed to verify whether, once all of the remodeling processes are completed, the newly formed tissue will acquire the more typical features of articular cartilage. Clinical Relevance: Our data provide important further information about the nature of the new cartilage tissue formed two years after autologous chondrocyte transplantation, allowing a better interpretation of the clinical and histological findings.

Besides the well-known activities of the prototypical inflammatory cytokines (IL-1beta, TNFalpha), a role for chemokines and their receptors in cartilage degradation in osteoarthritis has recently been reported. Human chondrocytes can produce CC and CXC chemokines and express chemokine receptors for both chemokine subfamilies. Engagement of these receptors can induce the release of matrix degrading enzymes such as matrix metalloproteinases 1, 3, and 13, and N-acetyl-beta-D-glucosaminidase. Furthermore GROalpha, a CXC chemokine acting on CXCR2, can activate an apoptotic pathway in chondrocytes that leads to chondrocyte cell death. These findings suggest that chemokines can act as an autocrine or paracrine loop on chondrocytes and can contribute to many pathophysiological patterns present in osteoarthritis. Chemokines and their downstream signaling pathways can be considered novel therapeutic targets in osteoarthritis.

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

The expression and functional activity of CXC chemokine receptors were evaluated in human osteoblasts (OB) obtained post-trauma from old donors compared to very young donors. It was found that CXCR1 and CXCR4 were only expressed by old but not young donors' cells. In contrast, CXCR3 and CXCR5 were expressed by both young and old donors. We functionally evaluated CXCR3/CXCL10 and CXCR5/CXCL13 receptor/ligand pairs by analysing cell proliferation and the release of N-acetyl-β-D-glucosaminidase (NAG), an enzyme that degrades glycosaminoglycans and hyaluronic acid. CXCL10 and CXCL13 induced a dose-dependent increase of cell proliferation in OB from young donors while cell proliferation of OB in old donors was not affected. By contrast, CXCL10 and CXCL13 induced a significantly higher NAG release in OB from old donors compared to young ones. These data demonstrate a significant age-dependent difference in the response of OB to CXCL10 and CXCL13 stimulation. These chemokines induce an inverse response of OB from old and young donors, which suggests a role of ageing in the modulation of cellular response of bone cells.

In the present study we analyse chemokine expression in the remodelling of subchondral bone in arthritis patients. Trabecular bone biopsies were tested by immunohistochemistry to identify interleukin (IL)-8, GRO-alpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression. Subsequently, we evaluated by immunoassay the effect of interferon (IFN)-gamma and IL-6 on chemokine production by osteoarthritis (OA), rheumatoid arthritis (RA) and post-traumatic (PT) patients' isolated osteoblasts (OB). OB constitutively produced in situ IL-8, GRO-alpha, MCP-1, RANTES and MIP-1alpha. MIP-1beta was positive only in mononuclear cells. In RA many of these chemokines were also produced by mononuclear cells. IFN-gamma significantly down-regulated IL-8 and up-regulated MCP-1 produced by OB from all patients tested, whereas it did not affect the other chemokines analysed. Moreover, IFN-gamma reduced IL-1beta-stimulated IL-8 production but significantly increased both MCP-1 and RANTES. Interestingly, IL-6 significantly downregulated IFN-gamma-induced MCP-1 production, that was significantly lower in OA compared to RA patients. OB expressed chemokines both in vivo and in vitro suggesting that these cells are primary effectors in the bone capable of regulating autocrine/paracrine circuits that affect bone remodelling in these diseases.

Objective To establish an immortalized normal human articular chondrocyte line which could be useful for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases. Design Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 (HPV-16) early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium supplemented with an insulin-containing serum substitute and seeding on a hyaluronan-based non-woven structured biomaterial. The expression of markers characteristic of cartilage was shown in the mRNA by reverse transcriptase-polymerase chain reaction. Immunohistological staining and Western blotting analysis were performed to evaluate type II collagen synthesis. Proteoglycans deposition was detected by Alcian Blue staining. A Field Emission In Lens Scanning Microscopy was used to look at the morphology of the immortalized cells at very high magnification. Results Normal human articular chondrocytes were efficiently transfected leading to the establishment of an immortalized cell line as confirmed by HPV-16 E7 mRNA and protein detection. These cells were able to re-express type II collagen both at mRNA and protein levels under the two defined cultured conditions we used, still maintaining type I collagen expression. Collagen IX mRNA was present only in early primary culture while collagen type X and aggrecan transcripts were always detected. Alcian Blue staining showed a proteoglycan-rich matrix production. The ultrastructural analysis of the immortalized cells revealed that their morphology strictly resembled that of normal chondrocytes. Conclusions The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation. Moreover, it appears to be a suitable model for pharmacological and toxicological studies related to rheumatic diseases relevant to humans.

Objective Fibroblasts play a major role in the development of systemic sclerosis (SSc), and the occurrence of serum autoantibodies reacting with fibroblast plasma membrane antigens in SSc has been reported. This study was undertaken to investigate whether IgG from SSc sera that react with human fibroblasts modulates the fibroblasts' function. Methods Sera from 69 patients with SSc (28 with limited cutaneous SSc [lcSSc] and 41 with diffuse cutaneous SSc [dcSSc]), 30 patients with sarcoidosis, and 50 matched healthy controls were examined. We evaluated antibody binding to human skin and lung fibroblasts by cell-based enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and flow cytometry. We further investigated the ability of purified IgG to modulate 1) intercellular adhesion molecule 1 (ICAM-1) expression, 2) U937 cell adhesion to fibroblasts, and 3) fibroblast steady-state messenger RNA (mRNA) levels of interleukin-1α (IL-1α), IL-β, and IL-6, and IL-6 protein production. Results Of 69 SSc sera tested by cell-based ELISA, 58% bound to normal skin and lung fibroblasts. The prevalence of binding was significantly higher in dcSSc than in lcSSc (P < 0.05). Only IgG from SSc sera that were positive for antifibroblast antibody (AFA) induced a dose-dependent up-regulation of ICAM-1 expression and IL-6 production, enhancement of U937 cell adhesion, and increased levels of IL-1α, IL-1β, and IL-6 mRNA in fibroblasts. Up-regulation of ICAM-1 mediated by AFA IgG was inhibited by the addition of IL-1 receptor antagonist, indicating an autocrine activation loop. Conclusion Our findings confirm the presence of AFAs in SSc sera and demonstrate, for the first time, that autoantibodies reacting with fibroblast surface molecules act as an extrinsic stimulus inducing fibroblast activation in vitro.

While numerous previous studies have investigated age-related changes of cytokine production, little is known about chemokines, the importance of which in regulating immune response is becoming increasingly evident. In this study, a group of healthy subjects over 90 years old is compared to a group of young subjects, we evaluated the ability of monocytes, T lymphocytes and NK cells: (1) to produce RANTES and MIP-1α, either in basal conditions or after stimulation with, respectively, LPS, anti-CD3 MoAb and IL-2; (2) to express the corresponding chemokine receptors (CCR1, CCR3, CCR5). We demonstrate that: (a) monocytes, T lymphocytes and NK cells spontaneously produced detectable amounts of chemokines, both in young and old subjects; (b) monocyte-dependent RANTES and MIP-1α production induced by LPS was up-regulated in nonagenarian subjects as anti-CD3-induced secretion from T cells; (c) RANTES and MIP-1α production by IL-2 stimulated NK cells was reduced in elderly subjects; (d) CCR1, CCR3 and CCR5 were widely expressed on monocytes, but less expressed on T lymphocytes and NK cells. The diversity within PBMC might reflect their different states of activation and/or responsiveness, influencing the ability to develop rapid innate and long-lasting adaptive immune responses.