A. Pinzano

Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis

Osteoarthritis is characterized by a gradual degradation of extracellular matrix, resulting from an excess of chondrocyte cell death, mainly due to an increase in apoptotis. Recent studies have revealed the essential role of HSP70 in protecting cells from stressful stimuli. Therefore, overexpressing HSP70 in chondrocytes could represent a good strategy to prevent extracellular matrix destruction. To this end, we have developed a vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to cell death induced by mono‐iodoacetate (MIA). To overcome the barrier‐effect of matrix, we investigated the efficacy of plasmid delivery by electroporation (EP) in rat patellar cartilage. Two days after EP, 50% of patellar chondrocytes were HSP/GFP+. After 3 months, long‐term expression of transgene was only depicted in the deep layer (20–30% positive cells). HSP70 overexpression inhibited the natural endochondral ossification in the deep layer, thus leading to a lesser decrease in chondrocyte distribution. Moreover, overexpression of HSP70, after a preventive EP transfer in rat patella, was sufficient to decrease the severity of osteoarthritis‐induced lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of HSP70, through EP delivery, could protect chondrocytes from cellular injuries and thus might be a novel chondroprotective modality in rat OA.—Grossin, L., Cournil‐Henrionnet, C., Pinzano, A., Gaborit, N., Dumas, D., Etienne, S., Stoltz, J. F., Terlain, B., Netter, P., Mir, L. M., Gillet, P. Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis. FASEB J. 20, 65–75 (2006)

Dose-Response of Superparamagnetic Iron Oxide Labeling on Mesenchymal Stem Cells Chondrogenic Differentiation: A Multi-Scale In Vitro Study

Aim The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues. Methods MSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity. Results No influence of various SPIO concentrations was noted on human bone marow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations. Conclusions This study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5–25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.

Nicorandil: from ulcer to fistula into adjacent organs

Nicorandil is an original vasodilatator used to control angina by decreasing cardiac preload and afterload. Since 1997, many reports of single or multiple nicorandil‐induced ulcerations have been published. To date, eight cases of nicorandil‐induced fistula into adjacent organs have been described. The pathogeneses of nicorandil‐induced ulceration and fistula into adjacent organs are not yet elucidated. The two main hepatic biotransformation pathways of nicorandil are denitration and reduction of the alkyl chain leading to nicotinamide and niconitic acid which merge into the endogenous pool of nicotinamide adenine dinucleotide/phosphate. This merging which is known as saturable, may contribute to a slow and abnormal distribution of nicotinamide and nicotinic acid out of the endogenous pool. Under these special conditions, providing these two molecules in situ, nicotinic acid associated with nicotinamide may ulcerate rather recent or maintained trauma. Ulcers and fistulae induced by nicorandil heal after withdrawal. Surgical intervention is unnecessary and inappropriate as it is ineffective and exacerbates morbidity. All practitioners should be correctly informed about these serious but preventable nicorandil side effects, which mostly occur in the elderly and fragile population. In the absence of corrective measures, withdrawal of this original and active drug should be considered.

169 EFFECTS OF SUPERPARAMAGNETIC IRON OXIDE LABELING ON CHONDROCYTE DIFFERENTIATION OF MESENCHYMAL STEM CELLS IN COLLAGEN SPONGE

Purpose: Stem cell therapy has emerged as a promising approach for orthopaedic treatment because of their potential ability of regenerating damaged tissues. One of the challenges is the ability to track implanted cells noninvasively following implantation. Several papers have addressed the topic of visualizing and tracking transplanted cells non-invasively using superparamagnetic iron oxide (SPIO) in magnetic resonance imaging (MRI). The aim of this study is to determine if SPIO internalization interferes with the chondrogenic differentiation ability of human mesenchymal stem cells (MSCs) seeded in collagen sponge biomaterials. Methods: Human bone marrow-derived mesenchymal stem cells (hMSCs) were expanded in monolayer and are first labeled with different SPIO concentrations between 0 and 200 μg/ml Fe. The endorem-labeled hMSCs were cultured into collagen sponge during 28 days with or without TGF-β1 to induce chondrogenic differentiation. Chondrogenic gene expression (coll 2, coll 1, coll 10, aggrecan, versican, COMP, sox 9, osteocalcin, alkaline phosphatase) were investigated by using real-time quantitative RT-PCR. Synthesized extracellular matrix inside sponge was assessed histologically (proteoglycan and collagen content, SPIO detection) and immunohistochemically (coll 1, coll 2). Results: TGF-β1 induced an increase of chondrogenic gene expression (col 2, aggrecan, Sox 9, COMP) and an abundant synthesized extracellular matrix inside sponge. SPIO labeling of MSC interfered with chondrogenesis: a dose-dependent decrease of gene expression such as aggrecan, Coll 1, coll 2, Sox 9, COMP was observed in SPIO labeled cells. Histologically, the quality and the quantity of synthesized matrix were affected by SPIO labeling in a dose dependant manner. The low concentration 12.5 μg/ml Fe induced a small decrease of proteoglycan and collagen 2 content. From 50 μg/ml Fe, a marked inhibition of chondrogenesis was observed: extracellular matrix was poor in proteoglycan and collagen contents. Conclusions: We observed a dose-dependent deleterious effect of SPIO concentration on the chondrogenic differentiation of human MSCs. The use of SPIO labeling to monitor cell-based therapies in MSC driven articular cartilage engineering requires very low SPIO concentration to minimize significant influence cell metabolism.