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Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 532 - 532
1 Nov 2011
Galois L Cournil-Henrionnet C Huselstein C Mainard D Bensoussan D Stoltz J Netter P Gillet P Watrin-Pinzano A
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Purpose of the study: Monolayer cultures of chondrocytes multiply and rapidly lose their chondrocyte phenotype, limiting their potential for tissue engineering. Mesenchymatous stem cells can preserve their phenotypic characteristics after several monolayer passages, offering a promising alternative for cartilage repair. The purpose of this work was to study the influence of transforming growth factor beta-1 (TGF-beta1) and bone morphogenic protein-2 (BMP2) and/or culture supplements (hyaluronic acid) on matrix synthesis and chondrocyte differentiation of human mesenchymatous stem cells (MSC) cultured on collagen sponges.

Material and methods: MSC were isolated from bone marrow harvested during hip arthroplsty. At the third passage in monolayer culture, the MSC were reseeded on collagen sponges and cultured in vitro for 28 days under seven differ conditions: insulin transferrin selenium (ITS), foetal calf serum (FCS), ITS+TGFbeta1, ITS+ hyaluronate, ITS+TGFbeta1+hyaluronate, ITS+TGFbeta1+BMP2, ITS +TGFbeta1+BMP2+hyaluronate. The phenotypic evolution was followed using the expression of different genes of interest with PCRq (collagen2, collagen1, collagen3, collagen10, agrecanne, versicanne, COMP, Sox9). Synthesis of matrix material was assessed histologically and immunohistochemically.

Results: Used alone, hyaluronic acid did not trigger chondrocyte differentiation of MSC. For the additives FCS, ITS, or hyaluronate, the synthesis of matrix material in the sponge was weak and poor in major constituents of cartilage. Conversely, the other conditions in presence of TGFbeta1±BMP2 induced important expression of collagen2, agrecanne and COMP as well as increased matrix synthesis with a strong content in proteoglycans and collagen.

Discussion: The usefulness of MSC is growing due to their pluripotent characteristics. The conditions leading to their differentiation into the chondrocyte phenotype remains a subject of discussion. Our results show the particular importance of TGFbeta1 in the process of differentiation.

Conclusion: Chondrogenic differentiation of MSC cultured in collagen sponges as well as the synthesis of the cartilaginous matrix requires the presence of TGFbeta1 in the culture medium and to a lesser extent BMP2. These results suggest the perspective of using MSC for guided cell therapy targeting cartilage.


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_I | Pages 171 - 171
1 Mar 2009
MAINARD D POTTIE P PRESLE N TERLAIN B GALOIS L LOEUILLE D NETTER P
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Objectives. To evaluate the contribution of leptin, an adipose-derived hormone, to the pathophysiology of osteoarthritis (OA), by determining leptin in both synovial fluid and cartilage specimens from human joints, and by investigating the effect on cartilage of intra-articular injection of leptin in rat.

Methods. Leptin levels were measured by enzyme-linked immunosorbent assay (ELISA) in synovial fluids sampled from OA patients undergoing either knee replacement surgery or knee arthroscopy. Besides, histological sections of articular cartilage and osteophytes obtained during surgery for total knee replacement, were graded using the Mankin score, and were immunostained using antibodies to leptin, TGF_ and IGF-1. For experimental studies, various doses of leptin (10, 30, 100 and 300μg) were injected into the rat knee joint. Tibial plateaus were collected and further processed for proteoglycan synthesis by radiolabeled sulfate incorporation, and for expression of leptin, its receptor (Ob-Rb), and growth factors by RT-PCR and immunohistochemistry.

Results. Leptin was found in synovial fluids from human OA-affected joints, and concentrations were correlated to Body Mass Index. A marked expression of the protein was seen in OA cartilage and in osteophytes, while few chondrocytes produced leptin in normal cartilage. Furthermore, the pattern and level of leptin expression were related to the grade of cartilage destruction, and paralleled those of growth factors (IGF-1 and TGFb-1). Animal studies showed that leptin strongly stimulated anabolic functions of chondrocytes, and induced the synthesis of IGF-1 and TGFb-1 in cartilage at both mRNA and protein levels.

Conclusion. These findings provide a new peripheral function to leptin as a key regulator of chondrocytes metabolism, and indicate that leptin may play an important role in the pathophysiology of OA.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_II | Pages 254 - 255
1 Jul 2008
MAINARD D DUMONT H PRESLE N TERLAIN L GALOIS L LOEUILLE D NETTER P POTTIE P
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Purpose of the study: This study was designed to assess the role of leptin in the development of osteoarthritis (OA) by searching for its presence in the synovial fluid (SF), tissues, and cartilage of osteoarthritic joints in humans and by observing the effect of intra-articular injections of leptin in the rat.

Material and methods: The leptin level in SF was measured (ELISA) in twenty patients (ten female, ten male, mean age 70 years). Presence of leptin, TGF beta and IGF1 in cartilage (and osteophytes) was detected by immunohistochemistry after histological evaluation (Mankin). In the rat, leptin was injected into the knee joint at the dose of 30 and 100 μg. After the immunohistological study, proteoglycan synthesis was assessed (S35 integration) as was the expression of leptin, TGF beta1 and IGF1 using RT-PCR.

Results: This study demonstrated for the first time the presence of leptin in synovial fluid (0.6–17.4 and 5.3–28.4 μg in male and female specimens respectively). There was a significant correlation with body mass index. Leptin was over expressed in chondrocytes of osteoarthritic cartilage and was correlated with the histological score (leptin not detecable in normal cartilage). IGF1 and TGF beta1 were expressed in osteoarthritic chondrocytes. The topographic distribution and the intensity of labeling varied with the histological score. There was a strong expression of TGF beta 1 only in osteophytes. In the rat, leptin stimulated anabolic functions of the chondrocyte: maximal effect at 30 μg (medial tibial plateau) and 100 μg (lateral tibial plateau). Leptin over expressed transcripts IGF 1 and TGF beta 1. This effect was confirmed at the protein level.

Discussion: Leptin is an adipocytokin which regulates food intake and energy expenditure at the hypothalamic level. A mechanical mechanism is the primary explanation of osteoarthritis in weight-bearing joints in obese patients. But leptin is also present specifically in non-weight-bearing joints in obese subjects. A biological factor is thus incriminated which might be leptin produced by adipose tissue. Leptin is overexpresssed in the cartilage of the osteoarthritic knee. This is in favor of a role for leptin in the pathogenesis of OA via synthesis of TGF beta 1 and IGF 1. This effect of leptin could explain the relationship between body mass index and the risk factor for osteoarthritis.


Orthopaedic Proceedings
Vol. 86-B, Issue SUPP_I | Pages 32 - 32
1 Jan 2004
Galois L Etienne S Grossin L Cournil C Netter P Minard D Gillet P
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Purpose: Section of the anterior cruciate ligament (ACL) is classically used to induce experimental joint degeneration in animal models (dog, rabbit, rat…), but the contribution of physical activity to the course of the cartilage damage observed in this model remains unknown. We studied the influence of moderate physical activity on the course of experimental knee joint degeneration induced by section of the ACL in the rat.

Material and methods: The right knee ACL was sectioned with and arthrotome in 60 male Wistar rats (180 g) under general anaesthesia. Full section of the ACL, performed with a fine lancet, was verified clinically by demonstrating anterior drawer. The non-operated knee served as a control. The rats were separated at random into two groups, with or without exercise. Exercise was calibrated on a treadmill running at constant speed (30 cm/s for 30 min, i.e. 15 km for 28 days). Rats were sacrificed on days 7, 14 and 28. Macroscopic inspection, histological analysis and immunohistochemistry tests (Caspase 3) were performed on each knee segment. NO was also assayed in the synovial fluid.

Results: No cartilage damage was observed in the non-operated knees after running 15 km. Marked synovitis was observed in the knees with a sectioned ACL starting on day 7, associated with fibrillary surface formations. The severity of the cartilage damage increased from day 14 to day 28, predominantly on the medial tibial plateau and to a lesser extent on the adjacent femoral condyle, in the weight-bearing zone. Damage was minimal on the patella. Chondrocyte apoptotic phenomena were also observed, reaching maximum on day 7 and sustained thereafter. Physical activity had a significant effect on these parameters showing an improvement in the macroscopic and histological lesions from day 14 to day 28, and improvement in chondrocyte apoptosis from day 7 to day 14 and to day 28.

Discussion: This novel work confirms the beneficial effect of moderate physical activity in an experimental joint degeneration rat model. Elsewhere, it has been well established experimentally that intense joint activity has a deleterious effect on chondral lesions after meniscectomy and/or section of the ACL. This unfavourable effect of intense physical activity has also been observed clinically in high-level athletes. Our experimental data suggest that moderate physical activity does not increase the risk of joint degeneration and could, under certain conditions, have a beneficial effect, as has been suggested by certain recent clinical data.