Didier Mainard

Poly(l-lysine) nanostructured particles for gene delivery and hormone stimulation

In this work, we designed replica particles based on poly (L-lysine) (PLL) polymers crosslinked via a homobifunctional linker to support coadsorption of a plasmid DNA and a peptide hormone for concurrent transfection and induction of a cellular function. PLL replica particles (PLL(RP)) were prepared by infiltrating polymer into mesoporous silica (MS) particles, crosslinking the adsorbed chains by using a homobifunctional crosslinker and finally removing the template particles. Moreover, we verified their cytotoxicity. Furthermore, based on this PLL(RP) gene delivery system, we simultaneously evaluated the melanin stimulation and gene expression in these cells by fluorescence microscopy. To further understand the bi-functionality, we labeled the SPT7pTL and PGA-alpha-MSH with YOYO-1 and Rhodamine, respectively, to follow its intracellular pathway by confocal microscopy. Our data suggests that the PLL(RP) is a promising vector for gene therapy and hormone stimulation.

Establishment of a Reliable Method for Direct Proteome Characterization of Human Articular Cartilage

Articular cartilage consists mainly of extracellular matrix, mostly made of collagens and proteoglycans. These macromolecules have so far impaired the detailed two-dimensional electrophoresis-based proteomic analysis of articular cartilage. Here we describe a method for selective protein extraction from cartilage, which excludes proteoglycans and collagen species, thus allowing direct profiling of the protein content of cartilage by two-dimensional electrophoresis. Consistent electrophoretic patterns of more than 600 protein states were reproducibly obtained after silver staining from 500 mg of human articular cartilage from joints with diverse pathologies. The extraction yield increased when the method was applied to a chondrosarcoma sample, consistent with selective extraction of cellular components. Nearly 200 of the most intensely stained protein spots were analyzed by MALDI-TOF mass spectrometry after trypsin digestion. They represented 127 different proteins with diverse functions. Our method provides a rapid, efficient, and pertinent alternative to previously proposed approaches for proteomic characterization of cartilage phenotypes. It will be useful for detecting protein expression patterns that relate pathophysiological processes of cartilaginous tissues such as osteoarthritis and chondrosarcoma.

Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion

Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.

Transfection ability and intracellular DNA pathway of nanostructured gene-delivery systems.

Considerable efforts have been devoted to the design of structured materials with functional properties. Polyelectrolyte multilayer films are now a well-established nanostructured concept with numerous potential applications, in particular as biomaterial coatings. This technique allows the preparation of nanostructured architectures exhibiting specific properties for cell-activation control and local drug delivery. In this study, we used a multilayered system made of poly-(l-lysine)/hyaluronic acid (PLL/HA) as a reservoir for active DNA complexes with nonviral gene-delivery vectors, PLL, beta-cyclodextrin (CD), and PLL-CD. When embedded into the multilayered films, the transfection efficiencies of the DNA complexes and the cell viability were improved. The highest transfection efficiency was obtained with the PLL-CD/plasmid DNA (pDNA) complexes. We found that this high transfection efficiency was related to an efficient internalization of the complexes in the cell cytoplasm and selected nuclei domains through a nonendocytotic pathway. For the first time, we report the intracellular pathway of the pDNA in complexes incorporated into the multilayered system.

MAGNETIC RESONANCE IMAGING OF NORMAL AND OSTEOARTHRITIC CARTILAGE

(MRI) has been slow to gain acceptance for cartilage evaluation because of its limited spatial resolution and because of the poor contrast between cartilage and adjacent structures. Continual improvement in gradient performance and coil design and the development of more efficient pulse sequences have overcome many of the early limitations of MIU. These improvements make possible high-resolution multiplanar and 3-dimensional (3-D) images with a wide variety of contrast. In this article, we present recent refinements in MRI of cartilage and offer a key for interpreting the wide range of images that are produced. Technical factors in MRI The pattern of cartilage as seen on MRT depends on spatial resolution and contrast. These factors are related to the choice of sequence and can be modified in various ways by the addition of pulses and contrast agents. In some cases, artifacts can greatly affect the quality of the images of hyaline cartilage.

Does Biolox Delta ceramic reduce the rate of component fractures in total hip replacement

UNLABELLED Biolox Delta ceramic has been optimized with nano-sized, yttria-stabilized tetragonal zirconium and strontium oxide to help limit cracking propagation. Although its mechanical properties are better than those of earlier generation ceramics, existing data on this material are limited, thus the goals of this study were to determine: 1) the remaining rate of implant fracture; 2) the ideal combination of head diameter and component position. Hypothesis. We hypothesized that the use of the ceramic composite Biolox Delta had reduced the risk of implant fracture. Materials and methods. The bibliographic search (in Pubmed database with the key words «ceramic fracture» and «total hip prosthesis ») identified 46 articles on fractures in third or fourth generation ceramic components, including 5 involving Biolox Delta. Manufacturers data and ANSM (Agence nationale de sécurité du médicament et des produits de santé) (National Agency for Safety of Drugs and Medical Products) reports were compared with the few clinical cases published in the literature. Results. According to the manufacturer (CeramTec GmbH, Plochingen, Germany), the use of Biolox Delta ceramic has reduced the rate of femoral head fractures to 0.003% compared to 0.021% with alumina ceramic. The fracture rate of liners has remained stable, at approximately 0.03%. The number of ANSM reports confirmed these tendencies. The rate of head component fractures decreases as the head diameter increases. The quality of impaction on the morse taper (cleanliness of the taper, insertion along the axis) plays an important role. Although it is generally only available for cup sizes above 50mm, a 36-mm head diameter seems to be optimal because it prevents impingement between the cup rim and the neck of the stem, without increasing micro-separation with larger diameters. Conclusion. Although Biolox Delta ceramic is more resistant to fractures than alumina ceramic, it can be fractured under suboptimal implantation conditions including edge loading. Its use requires the same precautions as other hard-on-hard bearings and requires special attention to cup position, insertion on or in morse tapers and adjustment of leg length. LEVEL OF EVIDENCE V experts opinion.

Smart Hybrid Materials Equipped by Nanoreservoirs of Therapeutics

Nanobiotechnology enables the emergence of entirely new classes of bioactive devices intended for targeted intracellular delivery for more efficacies and less toxicities. Among organic and inorganic approaches currently developed, controlled release from polymer matrices promises utmost clinical impact. Here, a unique nanotechnology strategy is used to entrap, protect, and stabilize therapeutic agents into polymer coatings acting as nanoreservoirs enrobing nanofibers of implantable membranes. Upon contact with cells, therapeutic agents become available through enzymatic degradation of the nanoreservoirs. As cells grow, divide, and infiltrate deeper into the porous membrane, they trigger slow and progressive release of therapeutic agents that, in turn, stimulate further cell proliferation. This constitutes the first instance of a smart living nanostructured hybrid membrane for regenerative medicine. The cell contact-dependent bioerodable nanoreservoirs described here will permit sustained release of drugs, genes, growth factors, etc., opening a general route to the design of sophisticated cell-therapy implants capable of robust and durable regeneration of a broad variety of tissues.

C-Propeptides of Procollagens Iα1 and II that Differentially Accumulate in Enchondromas versus Chondrosarcomas Regulate Tumor Cell Survival and Migration

Chondrogenic tumors that exhibit benign or malignant behaviors synthesize variable amounts of cartilage-like extracellular matrix. To define the regulators of these phenotypes, we performed a proteomic comparison of multiple human chondrogenic tumors, which revealed differential accumulation of the C-propeptides of procollagens Ialpha1 and II (PC1CP and PC2CP) in malignant versus benign tumors, respectively. Expression patterns of PC1CP correlated with levels of tumor vascularization, whereas expression patterns of PC2CP suggested its susceptibility to immobilization within the extracellular matrix. Prompted by these observations, we investigated the functions of recombinant PC1CP and PC2CP in the extracellular matrix in soluble or immobilized states. Each induced beta1 integrin-mediated chondrocyte adhesion by distinct domains and efficacies, suggesting that they initiated distinct signaling pathways. Indeed, immobilized PC2CP, but not PC1CP, induced apoptosis of primary chondrocytes and EAhy926 endothelial cells. In contrast, soluble PC1CP, but not PC2CP, induced the migration of EAhy926 cells and increased vascular endothelial growth factor (VEGF) and CXCR4 expression in chondrocytes. Soluble PC2CP also increased VEGF expression, but along with a more pronounced effect on CXCR4 and matrix metalloproteinase 13 expression. Our findings suggest that PC1CP favors angiogenesis and tumor progression, but that PC2CP acts in a more complex manner, exerting antitumor and antiangiogenic properties through apoptosis induction when immobilized, but progression and metastasis when soluble. In summary, the relative levels of PC1CP and PC2CP and their interactions within the extracellular matrix contribute to tumor progression, angiogenesis, and metastasis in chondrogenic tumors.

The reliability of the anterior pelvic plane for computer navigated acetabular component placement during total hip arthroplasty: Prospective study with the EOS imaging system

INTRODUCTION Computer navigated total hip arthroplasty is mostly based on the use of the anterior pelvic plane (APP) as a reference. EOS is a new imaging system that provides three-dimensional analysis of the pelvis in a functional position with a low dose of radiation. The aim of this study was to evaluate the reliability of the APP for placement of the cup during computer navigated THA using EOS. HYPOTHESIS The reliability of the APP is limited for the placement of the acetabular cup during computer navigated THA. MATERIALS AND METHODS This was a prospective monocentric study using the EOS imaging system evaluating 44 patients in the standing position three months after computer navigated THA (Orthopilot). Reproducibility of EOS measurements were analyzed using SterEOS software and the reliability of the navigation data for the position of the cup were assessed. RESULTS Intra and interobserver reproducibility of the measurements of the orientation of the cup by EOS were good with correlation coefficients above 93% and 95% and confidence intervals of less than ±5°. Mean cup inclination and anteversion were 41.3° and 20.9° and 44.3° and 29.5° respectively in operatively and post-operatively. The differences between measurements of operative cup inclination using computer assisted navigation and the post-operative EOS measurements were significant (P<0.05) with a correlation coefficient of less than 40%. DISCUSSION Our study confirms the lack of precision of the APP as a reference for positioning of the acetabular component, especially in relation to anteversion. Although for many years the APP was considered to be a global reference, in fact, it is subject to significant inter-individual variations and variations during changes in position. These factors, associated with the difficulty of determining the preoperative APP, explain the lack of reliability of this reference. Preoperative evaluation of the orientation of APP by EOS and its integration into the navigation system could help the operator position these components. LEVEL OF EVIDENCE Level III Prospective diagnostic case controlled study.

Influence of trauma induced by judo practice on postural control

Although high‐level sports develop specific physiological and sensorimotor abilities involved in balance control, they also increase the risk of injuries. The influence on postural control of lower limb trauma induced by judo practice was examined in healthy and previously injured judoists (PIJ). During static and dynamic tests, performed with or without vision, PIJ achieved the best performances in maintaining proper balance, except for those with the most severe antecedents of trauma. The severity of the pathology appeared to be the most important parameter prejudicial to balance control while the location, frequency and diversity of trauma had only a modest impact. This shows that PIJ develop excellent sensory and cognitive adaptation abilities to constraints involving new patterns of compensation and of body scheme.