G. Lisignoli

Chemokine expression by subchondral bone marrow stromal cells isolated from osteoarthritis (OA) and rheumatoid arthritis (RA) patients

We analysed the spontaneous and cytokine‐stimulated production and expression in vitro of IL‐8, GROα, MCP‐1, RANTES, MIP‐1α, MIP‐1β, by subchondral bone marrow stromal cells (BMSC) isolated from RA, OA, post‐traumatic (PT) patients and normal donors (ND). BMSC were cultured in vitro in the presence or absence of IL‐1β and tumour necrosis factor‐alpha (TNF‐α), and assessed for chemokine production, expression and immunolocalization. BMSC from different sources constitutively released MCP‐1, GROα and IL‐8, but not MIP‐1α or MIP‐1β, while BMSC from ND constitutively released only IL‐8 and MCP‐1. IL‐8, GROα and RANTES production in basal conditions was significantly higher in RA patients than in ND. RANTES production was also higher in OA and RA than in PT patients. The combination of TNF‐α and IL‐1β synergistically increased the production of all chemokines tested except for RANTES. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) demonstrated that all chemokines not detectable in the supernatants were expressed at the mRNA level. Chemokine immunostaining was localized around the nuclei. This work demonstrates that BMSC from subchondral bone produce chemokines and indicates that these cells could actively participate in the mechanisms directly or indirectly causing cartilage destruction and bone remodelling.

Osteoblasts and stromal cells isolated from femora in rheumatoid arthritis (RA) and osteoarthritis (OA) patients express IL-11, leukaemia inhibitory factor and oncostatin M

We investigated both in vitro and ex vivo the role of mature osteoblasts (OB) and bone marrow stromal cells (BMSC) in RA and OA by analysing the expression of the following IL‐6‐type cytokines: IL‐11, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and IL‐6. OB and BMSC were isolated from femora of RA, OA and post‐traumatic (PT) patients, cultured in vitro in the presence or absence of IL‐1β and tumour necrosis factor‐alpha (TNF‐α), and assessed for the production and mRNA expression of IL‐6‐type cytokines. Trabecular bone biopsies were obtained from the inner portions of femoral heads and used for cytokine in situ immunostaining. Cultured OB and BMSC from different patients constitutively secreted IL‐11 and IL‐6 but not OSM. LIF was secreted only by BMSC, at very low levels. Interestingly, IL‐11 basal production was significantly higher in BMSC than in OB in all three groups tested. IL‐1β and TNF‐α strongly stimulated IL‐6‐type cytokine release (except for OSM) by both OB and BMSC. OSM was expressed only at mRNA levels in all groups studied. Cytokine immunostaining on bone biopsies confirmed the data obtained on cultured cells: IL‐11, IL‐6 and LIF proteins were detected both in mesenchymal (BMSC and OB) and mononuclear cells; OSM was found only in mononuclear cells. These data demonstrate that IL‐6‐type cytokines are constitutively expressed in the bone compartment in RA, OA and PT patients and can be secreted by bone cells at different stages of differentiation (BMSC and OB). This suggests that these cytokines may be involved in the mechanisms of bone remodelling in OA and RA.

Mechano-functional assessment of human mesenchymal stem cells grown in three-dimensional hyaluronan-based scaffolds for cartilage tissue engineering

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.

A multifaced DNA ploidy analysis to determine ovarian carcinoma aggressiveness

A microfluorometric DNA study was conducted on isolated cells from 64 fragments of ovarian epithelial tumors. Data analysis considered the DNA content of the main stemline and the prevalence of cells in the different compartments of the DNA ploidy histogram in relation to the mitotic activity index and the histologic architecture. Main stemlines were found remarkably stable in different parts of the same tumor. Peridiploid (1.8c to 2.2c) stemlines were found both in well‐differentiated and in poorly differentiated tumors. However, low aneuploid (2.2c to 3.0c) stemlines were mostly found in nonsolid tumors and high ploidy (3.2 to 5.04 c) stemlines were prevalent in solid tumors. A comparative analysis between DNA ploidy parameters and mitotic activity was useful to evaluate the expanding modalities of neoplastic cell populations. This analysis revealed that several tumors accumulate an excessive amount of heteroploid cells and others an excessive amount of G2 cells. Yet, most neoplastic cell populations vary from slow to rapidly growing patterns without relevant abnormalities in their ploidy graphic profiles. No relationship was found between stemline ploidy and histologic architecture compared with expanding modalities. These findings indicate that multifaced criteria combining features such as main ploidy, graphic profile, mitotic rate, and histologic architecture measured on one or more microsamples of the same tumor may help to objectively estimate the aggressiveness reached by the neoplastic cells at the time of clinical presentation.

Lack of anti-inflammatory and anti-catabolic effects on basal inflamed osteoarthritic chondrocytes or synoviocytes by adipose stem cell-conditioned medium.

OBJECTIVE To define whether good manufacturing practice (GMP)-clinical grade adipose stem cell (ASC)-derived conditioned medium (CM) is as effective as GMP-ASC in modulating inflammatory and catabolic factors released by both osteoarthritis (OA) chondrocytes or synoviocytes. METHODS OA chondrocytes and synoviocytes were treated with ASC-CM or co-cultured with ASC. Inflammatory factors (IL6, CXCL1/GROα,CXCL8/IL8, CCL2/MCP-1, CCL3/MIP-1α and CCL5/RANTES) and proteinases, such as metalloproteinase (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS4, ADAMTS5) and their tissue metalloproteinase inhibitors (TIMP1, TIMP3) were evaluated by qRT-PCR or immunoassays. The involvement of prostaglandin E2 (PGE2) was also analyzed. RESULTS Most ASC-CM ratios tested did not decrease IL6, CCL2/MCP-1, CCL3/MIP1-α, CCL5/RANTES on basal inflamed chondrocytes or synoviocytes in contrast to what we found using ASC in co-culture. CXCL8/IL8 and CXCL1/GROα were not decreased by ASC-CM on synoviocytes but were only partially reduced on chondrocytes. Moreover, ASC-CM was less efficient both on basal inflamed OA chondrocytes and synoviocytes in reducing proteinases, such as MMP13, ADAMTS4, ADAMTS5 and increasing TIMP1 and TIMP3 compared to ASC in co-culture. The different ratios of ASC-CM contain lower amounts of PGE2 which were not sufficient to reduce inflammatory factors. CONCLUSIONS These data show that ASC-CM has a limited ability to decrease inflammatory and proteinases factors produced by OA chondrocytes or synoviocytes. ASC-CM is not sufficient to recapitulate the beneficial effect demonstrated using ASC in co-culture with inflamed OA chondrocytes and synoviocytes and shows that their use in clinical trials is fundamental to counteract OA progression.