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Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_II | Pages 253 - 254
1 Jul 2008
POTIER E FERREIRA E BETTON O MEUNIER A SEDEL L PETITE H
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Purpose of the study: Cell therapy proposes to fill gaps left by bone stock loss using osteocompetent cells (mesenchymatous stem cells, MSC). Preclinical results have been promising but still require improvement particularly concerning stress to the MSC during in vivo implantation. Stress results from sudden transfer i) from oxygen medium (21% O2) to a hypoxic medium (0–5% O2 because O2 diffusion is limited to 200 mm from a blood vessel), ii) a cell support to an osteoconductor support, et iii) a rich medium (fecal calf serum, FCS) to a medium with a limited supply of nutrients, hormones and growth factors diffusing from the environing biological fluids. The purpose of this study was to evaluate in vitro the impact of these different factors on MSC survival.

Material and methods: Human MSC(hMSC) harvested from bone marrow (n= 5 donors) and sheep MSC (sMSC) obtained with a preclinical model (n = 5 animal donors) were exposed for 48 h(hMSC) or 72h (sMSC) to the following transfers: i) rich medium (10% FCS) to poor medium (1% FCS), ii) plastic support to osteo-conductor supports (alumina, calcium carbonate), and iii) oxygen medium (21% O2) to hypoxic medium (6% O2). sMSC were also exposed to prolonged hypoxia (48–120h). Cell death was determined using image analysis after live/dead cell staining.

Results: The results demonstrated that MSC are: i) sensitive to a decrease from 10% to 0% FCS; 14% death of hMSC and 17% death of sMSC), ii) sensitive to transfer onto osteoconductor supports (sMSC on calcium carbonate: 23%), iii) very sensitive to prolonged hypoxia (120h) when combined with decreased FCS (sMSC: 23%; hMSC: 98%). A complementary study on the influence of hypoxia on differentiation properties of surviving sMSC is under way.

Conclusion: If the in vivo results concord with the in vitro results, i.e. if massive cell death is observed 4 days after implantation due to hypoxia, the current transplantation conditions will have to be revisited. Acceleration of neovascularization of in vivo implants which would shorten the period of hypoxia should allow better survival of implanted sMSC.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_II | Pages 253 - 253
1 Jul 2008
DEGAT M DUBREUCQ G MEUNIER A DAHRI-CORREIA L PETITE H SEDEL L LOGEART-AVRAMOGLOU D
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Purpose of the study: Bone morphogenetic proteins (BMPs) are osteoinducing proteins which play a primordial role in bone repair. To obtain optimal mineralization in vivo, high doses of heparin binding growth factor must be used. Studies have demonstrated that functionalized dextranes (FD) present affinity for heparin binding growth factor. We studied the capacity of dextrane derivatives to interact with BMP-2 and potentialize its biological activity in vitro.

Material and methods: Different soluble FD were obtained by random substitution of carbosymethyl (CM), benzylamide (B) and sulfate (Su) groups on native dextrane chains. Gel electrophoresis was used to study the affinity of the anionic FDs for BMP-2. The effect of polymers on osteoinduction activity of BMP-2 was evaluated by histochemistry. ALP (an early marker) synthesized by mypoblasts C2C12 were dosed seven days after injection in presence of BMP-2 associated or not with polymers. IN addition, expression of osteocalcin (late marker) was quantified by RT-PCR.

Results: Electrophoresis demonstrated that DMCB and DMCBSu interacted with BMP-2. These interactions appeared to increase with B level but decreased with Su level. We worked with FD1, a DMCB with a high affinity for BMP-2. The ALP activity was clearly potentialized when BMP-2 was associated with heparin and even better with FD1. Expression of osteocalcin was also amplified with the FD1-BMP-2 association. The influence on the biological activity of BMP-2 of FD, presenting different degrees of substitution, was also tested. Only FDs containing a high concentration of B expressed affinity for BMP-2, potentializing the biological activity of the protein.

Discussion: Dextanes functionalized with a high rate of benzylamide substitution interact with BMP-2 while sulfate substitution limits such interaction. Only FDS which interact with BMP-2 can potentialize the protein’s biological activity in vitro. Two hypotheses can be put forward: i) FD presents BMP-2 to its receptor cell, ii) FD protects BMP-2 from proteolytic degradation or capture by antagonists. The capacity of FD1 to potentialize the biological activity of BMP-2 could be a way of reducing the quantity of growth factor needed for optimal bone repair.


Orthopaedic Proceedings
Vol. 86-B, Issue SUPP_I | Pages 47 - 47
1 Jan 2004
Hannouche D Petite H Meunier A Sedel L Vacanti J
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Purpose: Tissue engineering offers new therapeutic perspectives with the possibility of producing cartilage tissue for a large number of patients. These structures are composed of an absorbable synthetic support and competent cells. Two types of cells can be proposed: articular chondrocytes harvested from the peripheral part of the joint, or mesenchymatous stem cells (MSC) present in the bone marrow and possessing chondrogenic potential. The purpose of this study was to determine the optimal cell source and the best supporting material for in vitro production of cartilage.

Material and methods: Isolated rabbit MSC were harvested and amplified with cell culture for 21 days. After this period, 20–40 million cells/ml were combined with polyglycolic acid sponges (3 types of sponges 1x1x0.2 cm2) and cultured in TGFß-enriched medium under specific dynamic conditions allowing gas exchange. The tissue obtained was compared with structures of identical size obtained with differentiated chondrocytes harvested from the same animals. The study included a histological analysis and immunohistochemistry for type I, II, and X collagen and biochemistry for DNA content, glycosaminoglycanes (GAG) and type II collagen.

Results: After 3 weeks in culture, the composites obtained with MSC preserved their size and had the white pearly aspect of hyalin cartilage. The histological analysis and immunohistochemistry tests for type II collagen confirmed the presence of a cartilaginous matrix throughout the thickness of the fragments. The GAG and type II collagen contents were significantly higher with MSC compared with chondrocytes, irrespective of the supporting material.

Discussion: This study demonstrated that cartilaginous tissue fragments can be obtained with MSC cultured on PGA supporting material under very specific conditions. Use of these cells offers the advantage of easy harvesting followed by in vitro amplification, and thus less harvesting morbidity. Complementary studies are needed to evaluate the behaviour of these living materials after implantation in the articulation.


Orthopaedic Proceedings
Vol. 86-B, Issue SUPP_I | Pages 50 - 50
1 Jan 2004
Hannouche D Nizard R Meunier A Bizot P Sedel L
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Purpose: During revision of alumina-on-alumina prostheses, reimplantation of a ceramic head on the former stem raises the risk of head fracture. Furthermore, serious deformation of the metal head has been reported after failure of ceramic-on-ceramic prostheses. The purpose of this retrospective study was to evaluate the incidence of these complications and analyse clinical and radiological results of revision for alumina-on-alumina total hip arthroplasties.

Material and methods: This consectutive series included 107 patients (74 women and 33 men, mean age 65 years, age range 32–91 years) who underwent 118 revision arthroplasties from 1976 to 1997. The cause in the majority of the cases was isolated non-infected cup loosening (80%) or ceramic implant fracture (4%). There were 72 cemented alumina cups, 38 screwed cups, six press-fit metal-backed cups, and two massive alumina impacted cups. Cavitary acetabular osteolysis was present in 67% of the cases. The cup was replaced alone in 94 cases (80%). The implanted cup was a polyethylene cup in 93 cases and an alumina cup in 25. When the femoral stem was left in place, a new ceramic head was implanted in 49 cases (39 alumina, 10 zircone). The alumina head was replaced by a metal head in 18 cases.

Results: Four early dislocations and two infections were noted. Mean follow-up after revision surgery was 67 months. Fourteen patients were lost to follow-up. Sixteen patients required revision surgery, ten of them for repeated cup loosening. At last follow-up (mean 61 months), there were no failures among the 49 ceramic heads implanted on a stem left in place. No revisions were required for polyethylene cup wear or metal head wear. In all, 72 hips were evaluated at a minimum two years follow-up. The functional score at last follow-up was 15.2±3.5. Seven-year actuarial survival was 95.5% taking revision of non-infected revision as the endpoint.

Discussion and conclusion: Analysis of the results shows that acetabular loosening is the main cause of revision of alumina-on-alumina total hip arthroplasties. The femoral stem was loosened in only nine cases and could be preserved in 80% of the cases. Replacement of an alumina-on-alumina couple with a metal-on-polyethylene couple does not appear to be a contraindication in this series.