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Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_6 | Pages 128 - 128
1 Jul 2020
Teissier V Hamadouche M Bensidhoum M Petite H
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Polyethylene wear-debris induced inflammatory osteolysis is known as the main cause of aseptic loosening and long term revision total hip arthroplasty. Although recent reports suggest that antioxidant impregnated ultra-high molecular weight polyethylene (UHMWPE) wear-debris have reduce the osteolytic potential in vivo when compared to virgin UHMWPE, little is known about if and/or how PE rate of oxidation affects osteolysis in vivo. We hypothesized that oxidized UHMWPE particles would cause more inflammatory osteolysis in a murine calvarial bone model when compared to virgin UHMWPE.

Male C57BL/6 eight weeks old received equal amount of particulate debris overlaying the calvarium of (n=12/group): sham treatment (no particles), 2mg (6,75×107 particles/mg) of endotoxin-free UHMWPE particles (PE) or of endotoxin-free highly oxidized-UHMWPE (OX) particles. In vivo osteolysis was assessed using high resolution micro-CT and inflammation with L-012 probe dependent luminescence. At day 10, calvarial bone was examined using high resolution micro-CT, histomorphometric, immunohistochemistry analyses and qRT-PCR to assess OPG, RANK, RANK-L, IL-10, IL-4, IL-1b and TRAP genes expression using the protocol defined by individual TaqManTM Gene Expression Assays Protocol (Applied Biosystems).

In vivo inflammation was significantly higher in the OX (1.60E+06 ± 8.28E+05 photons/s/cm2) versus PE (8.48E+05 ± 3.67E+05) group (p=0.01). Although there was a statistically significant difference between sham (−0.27% ± 2.55%) and implanted (PE: −9.7% ± 1.97%, and OX: − 8.38% ± 1.98%) groups with regards to bone resorption (p=0.02), this difference was not significant between OX and PE (p = 0.14). There was no significant difference between groups regarding PCR analyses for OPG, RANK, RANK-L, IL-10, IL-4, IL-1b and TRAP (p = 0.6, 0.7, 0.1, 0.6, 0.3, 0.4, 0.7 respectively). Bone volume density was significantly decreased in PE (13.3%±1.2%) and OX (12.2%±1.2%) groups when compared to sham (15%±0.9%) (p < 0 .05). Histomorphometric analyses showed a significantly decreased Bone Thickness/Tissue Thickness ratio in the implanted group (0.41±0.01 mm and 0.43±0.01 mm) compared to sham group (0.69± 0.01) (p < 0 .001). However, there were no significant difference between OX and PE (p = 0.2).

Our findings suggest that oxidized UHMWPE particles display increased inflammatory potential. Results were not significant regarding in vivo or ex vivo osteolysis. As antioxidant-diffused UHMWPE induce less inflammation activity in vivo, the mechanism by which they cause reduced osteolysis requires further investigation.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_8 | Pages 83 - 83
1 Apr 2017
Gindraux F Oudina K Nallet A de Billy B Petite H Obert L
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Previous clinical studies have shown the efficacy of a foreign body-induced membrane combined with bone autograft for the reconstruction of traumatologic or pathologic large bone defects or, bone non union. This membrane, rich in mesenchymal stromal cells (MSC), avoids bone autograft resorption and promotes consolidation by revascularisation of the bone and secretion of growth factors. Reconstruction requires two different surgical stages: firstly, insertion of a cement spacer in the defect, and secondly, removal of the spacer, preservation of the foreign body-induced membrane and filling of the cavity by bone autograft. The optimal time to perform the second surgical stage remains unclear.

So, we aimed to correlate bone healing and, phenotype and function of cells isolated from the induced membrane, in patients whose second surgery was performed on average after 6 months (i.e. beyond the recommended time of one month). Cell phenotype was determined by flow cytometry and cell function by: alkaline Phosphatase enzyme activity, secretion of calcium and von Kossa staining. Second, using histological and immunohistochemistry studies, we aimed to determine the nature and function of induced membrane over time. Seven patients were included with their consent.

Results showed Treated patients achieved in all cases bone union (except for one patient) and in in vitro and histology and immunohistochemistry gave some indications which need to be completed in the future. First, patient age seemed to be an indicator of bone union speed and recurrent infection, appeared to influence in vitro MSC osteogenic potential and induced membrane structure. Second, we reported, in bone repair situation, the commitment over time in osteogenic lineage of a surprising multipotent tissue (induced membrane) able of vascularisation/ osteogenesis/ chondrogenesis at a precocious time. Finally, best time to perform the second stage (one month) could be easily exceeded since bone union occurred even at very late times.


Bone & Joint Research
Vol. 6, Issue 4 | Pages 208 - 215
1 Apr 2017
Decambron A Manassero M Bensidhoum M Lecuelle B Logeart-Avramoglou D Petite H Viateau V

Objectives

To compare the therapeutic potential of tissue-engineered constructs (TECs) combining mesenchymal stem cells (MSCs) and coral granules from either Acropora or Porites to repair large bone defects.

Materials and Methods

Bone marrow-derived, autologous MSCs were seeded on Acropora or Porites coral granules in a perfusion bioreactor. Acropora-TECs (n = 7), Porites-TECs (n = 6) and bone autografts (n = 2) were then implanted into 25 mm long metatarsal diaphyseal defects in sheep. Bimonthly radiographic follow-up was completed until killing four months post-operatively. Explants were subsequently processed for microCT and histology to assess bone formation and coral bioresorption. Statistical analyses comprised Mann-Whitney, t-test and Kruskal–Wallis tests. Data were expressed as mean and standard deviation.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_1 | Pages 114 - 114
1 Jan 2017
Decambron A Fournet A Manassero M Bensidhoum M Logeart-Avramoglou D Petite H Viateau V
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Bone tissue engineering constructs (BTEC) combining natural resorbable osteoconductive scaffolds and mesenchymal stem cells (MSCs) have given promising results to repair critical size bone defect. Yet, results remain inconsistent. Adjonction of an osteoinductive factor to these BTEC, such as rh-BMP-2, to improve bone healing, seems to be a relevant strategy. However, currently supraphysiological dose of this protein are used and can lead to adverse effects such as inflammation, ectopic bone and/or bone cyst formation. Interestingly, in a preliminary study conducted in ectopic site in a murine model, a synergistic effect on bone formation was observed only when a low dose of rh-BMP-2 was associated with MSCs-seeded coral scaffolds but not with a high dose.

The objective of the study was then to evaluate a BTEC combining coral scaffold, MSCs and a low dose of rh-BMP-2 in a large animal model of clinical relevance.

Sixteen sheep were used for this study.

MSCs were isolated from an aspirate of bone marrow harvested from the iliac crest of each sheep receiving BTEC with MSCs, cultivated and seeded on Acroporacoral scaffolds one week before implantation.

Rh-BMP-2, used at two different doses (low dose: 68μg/defect and high dose: 680μg/defect), was diluted and absorbed on Acroporacoral scaffold one day before implantation.

Metatarsal segmental bone defects (25 mm) were made in the left metatarsal bone of the sheep, stabilized by plate fixation, and filled with Acroporacoral scaffolds loaded with either (i) MSCs and a low dose of rh-BMP-2 (Group 1;n=6), (ii) a low dose of rh-BMP-2 (Group 2;n=5), (iii) a high dose of rh-BMP-2 (Group 3;n=5). Standard radiographs were taken after each surgery and each month until sheep sacrifice, 4 months postoperatively. Bone healing and scaffold resorption were assessed by micro-computed-tomography (μCT) and histomorphometry. Results were compared to a historical control group in which coral scaffolds were loaded with MSCs.

Bone volumes (BV) evaluated by μCT and bone surfaces (BS) evaluated by histomorphometry did not differ between groups (BV: 1914±870, 1737±841, 1894±1028 and 1835±1342 mm3; BS: 25,41±14,25, 19,85±8,31, 25,54±16,98 and 26,08±22,52 %; groups 1, 2, 3 and control respectively); however, an higher bone union was observed in group 1 compared to the others (3, 1, 2 and 2 sheep with bone union in groups 1, 2, 3 and control respectively). No histological abnormalities were observed in any group. Coral resorption was almost complete in all specimens. No significant difference in coral volumes and coral surfaces was observed between groups. A trend towards a higher variability in coral resorption was noted in group 1 compared to the others.

There seems to be a benefit to associate low dose of rh-BMP-2 with MSCs-seeded coral scaffolds as this strategy allowed an increase of bone unions in our model. Yet, results remain inconsistent. Although, defective coupling between scaffold resorption and bone formation impaired bone healing in some animals, adjunction of rh-BMP-2 (even at low dose) to CSMs loaded construct is a promising strategy for bone tissue engineering.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 243 - 243
1 Jul 2014
Decambron A Manassero M Bensidhoum M Petite H Viateau V
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Summary

MSCs could promote bone regeneration in sheep when loaded on natural fully-resorbable scaffolds, but results are highly variable. Improving the ultimate performance of cell-containing constructs cannot be limited to the decreased rate of scaffold resorption.

Introduction. Tissue constructs containing mesenchymal stem cells (MSCs) are an appealing strategy for repairing massive segmental bone defects. However, their therapeutic effectiveness does not match that of autologous bone grafts; among the failure reasons the scaffold resorbability has been identified as a critical feature for achieving bone regeneration. In the present study, the osteogenic potential of 2 constructs obtained by expanding in a bioreactor autologous MSCs onto granules of Acropora or Porites coral, natural fully-resorbable scaffolds, was compared.

Materials and methods

15 sheep underwent a 25 mm long metatarsal ostectomy stabilised with a 3.5 DCP plate. Bone defects were replaced with (i) MSCs-Acropora constructs (n=7), (ii) MSCs-Porites constructs (n=6), (iii) autograft (n=2). Animals were sacrificed 4 months later and bone healing and coral resorption was documented by radiographic, histologic and microCT studies.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 117 - 117
1 Jul 2014
Deschepper M Paquet J Petite H
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Introduction

The use of mesenchymal stem cells in regenerative medicine remains a promising approach due to the ability of these cells to differentiate into a variety of cell types of mesodermal lineage. Today, however, it is not clear whether long-term differentiation of MSCs is necessary or alternatively whether the benefits of MSCs can be conferred by transitory paracrine effects (via secreted chemical compounds). Human MSCs secrete a broad variety of cytokines, chemokines and growth factors that may potentially be involved in tissue repair. Nevertheless, hMSCs secretome profile is closely related to cells biological and chemical environment (pO2, inflammation, nutrients disponibility…). In the context of stem-cell-based regenerative medicine, upon implantation, hMSC are exposed to stresses such as ischemia, oxidative stress and inflammatory mediators. Knowledge of the paracrine properties of stem cells under hypoxic conditions is essential for planning appropriate strategies that overcome the potential negative impacts of all levels of low oxygen content (from hypoxiato anoxia) leading to ischemia and tissue necrosis pertinent to MSC-based tissue engineered constructs. Since the beneficial effects of stem cells may be confered predominantly indirectly through paracrine mechanisms, the present study was designed to characterise the hMSC secretome and to assess its biological effects considering oxygen level and nutrients disponibility.

Methods

hMSCs were exposed in vitro either to sustain ischemic environment (pO2 ≤ 0.1%, serum deprived), to hypoxia (pO2 ≤ 0.1%, 5g/L glucose) or to normoxic conditions (pO2=21%). We used an hypoxic station (Biospherix, US) to ensure sustained hypoxia during 21 days. Levels of angiogenic, chemo-attractant, inflammatory and immunomodulative mediators were assessed in supernatants using the luminex technology (milliplex KIT, Millipore, USA). The chemo-attractant potential of conditioned media (CM) was assessed in vitro using Boyden chambers (BD, USA). To assess angiogenic potential of CM, HEPC were seeded on matrigel for 18 hours with CM obtained from hMSCs. Functionnality of secreted mediators was also assessed in vivo: briefly, CM media were lyophilyzed on collagen sponges and ectopically implanted in nude mice. Chemo-attraction and vascularization of the implants were determined using histological and CTscan analysis.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 238 - 238
1 Jul 2014
Deschepper M Paquet J Manassero M Logeart-Avramoglou D Bensidhoum M Petite H
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Summary

In this study, we challenged the current paradigm of human Mesenchymal Stem Cells survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to their survival.

Introduction

The survival of human mesenchymal stem cells (hMSCs) has elicited a great deal of interest, because it is relevant to the efficacy of engineered tissues. However, to date, hMSCs have not met this promise, in part due to the high death rate of cells upon transplantation. In this study, we challenged the current paradigm of hMSC survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to hMSC survival.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 70 - 70
1 Jul 2014
Manassero M Viateau V Decambron A Deschepper M Bensidhoum M Logeart D Petite H
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Summary

Despite similar, early and massive death, hMSCs promote bone formation which was higher in orthotopic than ectopic site suggesting a trophic effect of hMSCs. Ectopic implantation is suitable to evaluate cell survival, but assessment of bone formation requires orthotopic implantation

Introduction

Tissue constructs containing mesenchymal stem cells (MSCs) are appealing strategies for repairing large segmental bone defects but they do not allow consistent bone healing and early and massive MSCs death was identified as a cause of failure. However, little is known about cell survival in the clinical micro-environment encountered during bone healing process, whereas ectopic evaluation is well documented.

In vivo, luciferase-labelled human MSCs survival, within osteoconductive scaffold, was compared in orthotopic and ectopic locations, and bone formation ability of LF-hMSCs-Acropora constructs was evaluated. Interest and limits of each model were highlighted.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 191 - 191
1 Jul 2014
Sladkova M Manassero M Myrtil V Savari H Fall M Thomas D Bensidhoum M Logeart-Avramoglou D Petite H
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Introduction

The use of mesenchymal stem cells (MSCs) loaded on osteoconductive scaffolds has emerged as a potential new treatment of large bone defects but has generated marginally successful results in terms of new bone formation. It is supposed that MSC massive death post implantation is a major obstacle for the exhibition of their osteogenic potential. Yet, the very few studies conducted using primary human MSCs derived from bone marrow (hMSCs), a clinically pertinent cell source, did not demonstrate that cell survival is required for new bone formation. In order to elucidate whether cell survival is needed for hMSC to express their osteogenic potential, the present study examined in an ectopic mouse model the relationship between cell survival and osteogenic potential of hMSCs loaded onto osteoconductive scaffold.

Materials and Methods

hMSCs (106) were seeded on 40-mg calcium carbonate (Biocoral) particles (size: 610–1000 µm), wrapped in fibrin gel (Baxter), and implanted subcutaneously into immunodeficient (nu/nu) mice (n=8/group). The fate of implanted cells was analysed using the bioluminescence and immunohistochemistry. For this, hMSCs were transduced with Luc-GFP (Luciferase-Green fluorescent protein) lentiviral vectors prior to experimentation. Bone formation was analysed 8 weeks post implantation on both non-decalcified and decalcified samples.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 114 - 114
1 Jul 2014
Viateau V Manassero M Petite H Logeart-Avramoglou D Sladkova M Oudina K Bensidhoum M
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Short Summary

The present study demonstrated the feasibility of culturing a large number of standardised granular MSC-containing constructs in a packed bed/column bioreactor that can produce sheep MSC-containing constructs to repair critical-size bone defects in sheep model.

Introduction

Endogenous tissue regeneration mechanisms do not suffice to repair large segmental long-bone defects. Although autologous bone graft remains the gold standard for bone repair, the pertinent surgical technique is limited. Tissue constructs composed of MSCs seeded onto biocompatible scaffolds have been proposed for repairing bone defects and have been established in clinically-relevant animal models. Producing tissue constructs for healing bone defects of clinically-relevant volume requires a large number of cells to heal an approximately 3 cm segmental bone defect. For this reason, a major challenge is to expand cells from a bone marrow aspirate to a much larger, and sufficient, number of MSCs. In this respect, bioreactor systems which provide a reproducible and well-controlled three-dimensional (3D) environment suitable for either production of multiple or large size tissue constructs are attractive approaches to expand MSCs and obtain MSC-containing constructs of clinical grade. In these bioreactor systems, MSCs loaded onto scaffolds are exposed to fluid flow, a condition that provides both enhanced access to oxygen and nutrients as well as fluid-flow-driven mechanical stimulation to cells. The present study was to evaluate bioreactor containing autologous MSCs loaded on coral scaffolds to repair critical-size bone defects in sheep model.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 78 - 78
1 Jul 2014
Sudre L Cruel M Becquart P Hoc T Petite H Bensidhoum M
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Summary

Shear stress and hydrostatic effects on the hMSCs early mechano gene response were similar. For the same magnitude gene response, the hydrostatic compression (1.5×105 Pascal) is a 200000 times greater than the force exerted by shear stress (0.7 Pascal).

Introduction

In the lab, a perfusion bioreactor designed to automate the production of bone constructs was developed. The proof of concept was established in a large animal model of clinical relevance. The cells perfused in the bioreactor are likely to perceive 2 types of stresses: shear stress and hydrostatic pressure. Optimization of this bioreactor implies a better understanding of the effects of these forces on the cells in order to have better proliferation and differentiation. An understanding of the response of one cell layer submit to various strength is relevant. The primary objective of this study was to test the hypothesis that hMSCs have the fundamental ability to distinguish between different types of mechanical signals as evidenced by distinct gene expression. The effect of shear stress on one cell layer cultures of hMSCs will be evaluated using a commercially available system called Ibidi. For the hydrostatic pressure as there is no commercial device available, our group has developed a prototype capable of delivering a well-defined mechanical loading to cells in culture.

Validation of the techniques: In order to validate the systems (shear stress and cyclic pressure apparatus) used in this study, we have used an osteocytes-like cell line, MLO-Y4. When stimulated by a 30 minutes PFF at 7 dyn/cm2 or hydrostatic compression at 1.5 bar, cells responded by producing NO in the culture media

NO release after mechanical stimulation of hMSCs: hMSCs were subjected to increased PFF (7 to 42 dyn/cm2) for 30 minutes. This stimulation resulted in an increased release of NO in the media compared to non-stimulated cells (p<0.05). Interestingly the level of NO was maximal at 7 dyn/cm2 and decreased with higher flow rate. Similar observation was made after hMSCs stimulation by hydrostatic pressure for 30 minutes: a peak of NO release at 1.5 bar was observed

Early gene expression of known mechano-sensitive genes: Gene expression analysis immediately after stimulation (PFF or hydrostatic compression) was performed on a range of known mechano-sensitive genes: NOS2, PTGS2, PTGES, IER3 and EGR1. Immediately after stimulation by PFF at 7 dyn/cm2 or hydrostatic pressure at 1.5 bars, the expression of all the genes of interest appear to be up regulated in stimulated cells


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 118 - 118
1 Jul 2014
Logeart-Avramoglou D Monfoulet L Becquart P Pacard E Vandame K Bourguignon M Marchat D Petite H
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Summary

45S5 bioactive glass combined with hMSC did not permit de novo ectopic bone formation. Such absence of osteogenicity was most likely due to the alkalinization of the 45S5 microenvironment that affects adversely the osteogenic differentiation of stem/precursor cells.

Bone marrow stromal cells (BMSCs) are capable of bone formation and can promote the repair of osseous defects when implanted in appropriate scaffolds. The most promising biomaterials for application in bone tissue engineering (TE) are hydroxyapatite (HA), tricalcium phosphate (TCP), calcium carbonate (coral) ceramics or bioactive glasses (BG) because of their osteoconductive properties and ability to enhance bone formation. However, information regarding the osteogenic potential of hBMSCs in combination with BG scaffolds is strikingly lacking in the TE field. The present study focused on evaluating the osteogenicity of bone constructs prepared from particles of 45S5 BG combined with hBMSCs in comparison with biphasic HA/TCP or coral particles, in a mouse ectopic model.

The in vivo osteogenicity was then correlated with various aspects of the effects of the scaffold materials tested on hBMSCs functions pertinent to bone tissue formation. Particular attention was given to the pH in the microenvironment where the cells reside in TE constructs and its effect on the osteoblastic differentiation of hBMSCs. In vivo experiments evidenced that 45S5 BG constructs with hBMSCs failed to form ectopic bone. In contrast, the cell constructs prepared with either HA/TCP or coral ceramics displayed great and consistent capacity for the ectopic bone formation. The cytocompatibility of hBMSCs on BG material was addressed and no differences were evidenced between HA/TCP and coral substrates related to the adhesion of hBMSCs and their proliferation in vitro. The hBMSCs viability was even higher within the 45S5 BG-containing constructs compared to the other two types of material constructs tested both in vitro and in vivo. These findings indicated that the absence of de novo bone formation in the hBMSCs-containing 45S5 BG constructs was not the result of cytotoxic effects of the BG material.

The potential of osteogenic differentiation of hBMSCs cultured on material substrates was next addressed and the ALP activity of hBMSCs was significantly diminished when these cells were cultured on 45S5 BG as compared to either HA/TCP or coral substrates. Because BG materials are well-known for causing external alkalinisation, the pH was specifically measured in TE constructs. The pH inside the cell-containing BG constructs, measured ex vivo, was 8.0 (i.e. 0.4–0.5 units more alkaline than that measured in the coral- or HA/TCP-constructs). The impact of such external alkalinisation on the osteogenic differentiation of hBMSCs was assessed by culturing the cells over a wide range of alkaline pH. The hBMSCs expression of osteogenic markers, ALP activity and mineralization were not significantly affected at moderate external alkaline pH (≤ 7.90) but were dramatically inhibited at higher pH.

Altogether, these findings provided evidence that despite 45S5 BG are reported to be good osteoconductive materials, they are not necessarily good scaffolds for TE, most likely due to the alkalinization of the 45S5 microenvironment that affects adversely the osteogenic differentiation of precursor cells. Controlling the shifts of pH in the local engineered extracellular environment is a critical issue for the development of bioactive TE scaffolds.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVII | Pages 126 - 126
1 Sep 2012
Nich C Nich C Langlois J Marchadier A Vidal C Cohen-Solal M Petite H Hamadouche M
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Osteoporosis following ovariectomy has been suggested to modulate bone response to polyethylene wear debris. In this work, we evaluate the influence of estrogen deficiency on experimental particle-induced osteolysis. Polyethylene (PE) particles were implanted onto the calvaria of wild-type (WT), sham-ovariectomized (OVX), OVX mice and OVX mice supplemented with estrogen (OVX+E2) (12 mice per group). Sham-implanted mice served as internal controls. After 14 days, seven skulls per group were analyzed with a high-resolution micro-computed tomography (CT) and by histomorphometry, and for tartrate-specific alkaline phosphatase. Five calvariae per group were cultured for the assay of IL-1, IL-6, TNF- and RANKL secretion using quantitative ELISA. The expression of RANKL and OPG mRNA were evaluated using real-time PCR. As assessed by CT and by histomorphometry, PE particles induced an extensive bone resorption and an intense inflammatory reaction in WT, sham-OVX and OVX+E2 mice. In OVX mice group, these features appeared considerably attenuated. In WT, sham-OVX and OVX+E2 mice, PE particles induced an increase in serum IL-6, in TNF-and RANKL local concentrations, and resulted in a two-fold increase in RANKL/OPG mRNA ratio. Conversely, these parameters remained unchanged in OVX mice after PE implantation. The combination of two well-known bone resorptive mechanisms ultimately attenuated osteolytic response, suggesting a protective effect of estrogen deficiency on particle-induced osteolysis. This paradoxical phenomenon was associated with a downregulation of pro-resorptive cytokines. It is hypothesized that excessive inflammatory response was controlled, illustrated by the absence of increase of serum IL-6 in OVX mice after PE implantation.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVII | Pages 401 - 401
1 Sep 2012
Aurégan J Bérot M Magoariec H Hoc T Bégué T Hannouche D Zadegan F Petite H Bensidhoum M
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Introduction

Osteoporosis is a metabolic disease of the bone responsible for a loss of bone resistance and an increase in fracture risk. World Health Organization (WHO) estimations are about 6.3 millions of femoral neck fractures in the world by 2050. These estimations make osteoporosis a real problem in term of public health.

Knowledge in biological tissues mechanical behaviour and its evolution with age are important for the design of diagnosis and therapeutic tools. From the mechanical aspect, bone resistance is dependent on bone density, bone architecture and bone tissue quality. If the importance of bone density and bone architecture has been well explored, the bone tissue quality still remains unstudied because of the lack of biomechanical tools suitable for testing bone at this microscopic dimension.

Therefore the goal of this study is to estimate the osteoporotic cancellous bone tissue mechanical behaviour at its microscopic scale, using an approach coupling mechanical assays and digital reconstruction.

Materials and methods

The experimental study is based on cancellous bone tissue extracted from human femoral head. Forty 8mm diameters bone cylinders have been removed from femoral head explanted after a femoral neck fracture treated by arthroplasty. These cylinders have been submitted to a digitally controlled compressive trial. Before and after the trials, microscanner analyses with an 8 μm spatial resolution have been realized in order to determine the micro structural parameters. The cylinders have been rebuilt with the digital model-building in order to estimate the mechanical behaviour and the bone quality.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 532 - 533
1 Nov 2011
Nich C Marchadier A Sedel L Petite H Hamadouche M
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Purpose of the study: Oestrogen depletion leads to osteoclastic hyperactivity and subsequent postmenopausal osteoporosis. Little is known about interactions with bone absorption induced by wear particles from joint bearings. The purpose of this study was to evaluate bone response to polyethylene (PE) particles in a mouse model of oestrogen deficiency.

Material and methods: Particles of PE were implanted in the calvaria of seven non-ovariectomised mice and in seven ovariectomised mice (OVX). Fourteen mice were operated on without implantation of the particles (7 non-OVX and 7 OVX, control groups). The mice were sacrificed at two weeks. The crania were studied under a microscanner and histologically without decalcification.

Results: The microscanner showed that particles of PE induced a significant decrease in bone thickness in non-OVX mice (p=0.04), while the thickness remained unchanged in OVX mice who had received the particles (p=0.40). After implantation of the PE particles, the number of osteoclasts per mm of bone perimeter was 2.84±1.6 in the non-OVX mice and 1.74±1.3 in OVX mice (p=0.004). Compared with controls, the mean loss of bone was 12±10% in the non-OVX mice versus 4.7±0.9%in the OVX mice (p=0.004).

Discussion: The volume of osteolysis induced by PE particles was smaller in OVX mice compared with non-OVX mice.

Conclusion: These results suggest that a deficit in oestrogens has a protective effect against bone adsorption induced by PE particles.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 532 - 532
1 Nov 2011
Lesaichot V Sailhan F Leperlier D Viateau V Petite H
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Purpose of the study: The process of bone lengthening involves three phases: a latence period, distraction, then healing. The healing phase required stability maintained by an external fixator (EF) for 1.16 months/cm lengthening. This time exposes the patient to serious complications. The objective is to accelerate the healing phase in order to shorten the time the patient has to wear the EF. The effect of BMP on osteogenesis in distraction remains a controversial issue. This work was conducted to evaluate the benefit provided by rhBMP-2 for healing the regenerate bone after distraction.

Material and methods: Thirty-nine subadult male rabbits were selected at random. On day 0, a tibial osteotomy was performed followed by installation of a M101 EF. After the latency period of seven days, the distraction began at the rate of 0.5mm/12 h for 21 days. At day 28, at the end of distraction, a new operation was performed and three groups of 13 individuals were created at random. The first group received no material, the second a collagen type 1 sponge, and the third group a collagen type 1 sponge soaked in 100 μg/kg rhBMP-2. The animals were monitored with x-rays, absorptiometry and ultrasound for the qualitative and quantitative analysis. Histological and biomechanical analyses were performed at two months.

Results: Our complication rate was 41%. Qualitative analysis of the x-rays showed, in group 3, the development of more or less voluminous and dense, sometimes hypertrophic calluses. The progression curves of the bone mineral content showed higher values in group 3. The bone mineral content curves remained nevertheless parallel for the three groups. The calluses were thus denser in group 3 but with an early peak density. Groups 1 and 2 had equivalent radiographic and absorptiometric results. The statistical analysis of the imaging findings is ongoing. The histology and biomechanical exams are being performed.

Discussion: The preliminary results show that rhBMP-2 used early in the healing phase enables formation of more dense and hypertrophic calluses. rhBMP-2 does not acceleration the rate of callus formation but stimulates its mineralization. Use of a collagen sponge alone had no effect on healing. Analysis of the histological and mechanic properties observed in the three groups will provide a more precise description of the hypertrophic and strongly mineralized calluses.

Conclusion: Our early results show superior bone mineralization in the treated group.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_II | Pages 253 - 253
1 Jul 2008
FERREIRA E POTIER E MIR L PETITE H SEDEL L
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Purpose of the study: Several studies have demonstrated the usefulness of mesenchymatous stem cells (MSC) for cell therapy aimed at favoring bone tissue healing. Bone morphogenesis proteins (BMP) orient MSC towards osteoblastic differentiation. Since they are rapidly degraded in the organism, these proteins require a continuous release system to potentialize their biological activity in a controlled localized manner. We evaluated the usefulness of using the electroporation technique to insert a BMP transgene into the MSC of rats to enable sufficient transient expression of BMP genes to enable satisfactory bone healing. We first developed electroporation conditions for rat MSC and checked cell viability after the electric shock. Secondly, in order to obtain quantitative and/or temporal BMP expression, we tested the influence of different promoters on transcription actvity.

Material and methods: To determine the electroporation parameters, MCS were transfected with the pCMV-LacZ plasmid using two electric impulsions: a series of eight 100 impulsions/μs at high voltage (900-170V/cm) followed or not by a series of eight 12.5 ms low-voltage impulsions (60 V/cm). After determining the electroporation conditions, six plasmids carrying different promoters were electroporated.

Results: The best transfection rate in rat MSC was obtained with a series of 8 impulsions at 1500 V/cm. Before the electrical shock, the suspended rat MSC had to be incubated at ambient temperature to favor cell survival. Proliferation of electroporated cells was comparable to that of non electroporated cells. Surprisingly, addition of low-voltage pulses significantly decreased the efficacy of transfection. In addition, MSC transfected with the promoters GAPDH and beta-actin presented a beta-galactoside activity (at 48 h) superior to that obtained with the pCMV promoter.

Discussion: After optimization of these parameters, we demonstrated that MCS can be effectively transfected by electroporation. The following steps will be to check for long-term expression of beta-galactoside by electroporated MSC, transfection of MSC with plasmids or the BMP-2 gene controlled by these same promoters and monitoring promoter activity as a function of the stage of MSC differentiation.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_II | Pages 254 - 254
1 Jul 2008
VIATEAU V GUILLEMIN G CALANDO Y OUDINA K SEDEL L HANNOUCHE D PETITE H
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Purpose of the study: The objective of this study was to establish an experimental sheep model for a surgical procedure which has been clinically successful for repairing major loss of bone stock: the Masquelet technique.

Material and methods: A 25 mm bone defect was created in a metatarsal bone then filled with a cement filler. After six weeks, the cement was removed after opening the neoformed pseudosynovial membrane. The cavity was left empty in group 1 (n=6) or filled with a morcelized cancellous autograft harvested from the iliac crests in group 1 (n=6).

Results: The surgery was well tolerated in all animals which were able to used the injured limb the day after the operation. Radiographic images and histological findings 24 weeks after surgery demonstrated that healing had not been achieved in all of the animals in group 1. Inversely, healing was achieved in all animals in group 2 at 24 weeks. Immunohistochemistry of the neoformed pseudosynovial demonstrated :

an abundant vascular network,

presence of cells expressing transcription factor CBFA1,

very few inflammatory CD14+ cells (macrophages),

an extracellular matrix positive for type I collagen.

Conclusion: The sheep metatarsal model is a model of critical size with low morbidity. This model could be used to:

evaluate new therapeutic strategies for bone regeneration in conditions close to clinical situations,

study the role of the membrane in bone repair.

The presence of a pseudosynovial membrane might:

be a barrier against the diffusion of bone morphogenetic proteins outside the lesion and

potentially be a reservoir of stem and vascular cells which could be useful for new technologies.


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.