Christel Henrionnet

Evaluation of intra-articular delivery of hyaluronic acid functionalized biopolymeric nanoparticles in healthy rat knees.

The aim of this study is to evaluate the toxicity of nanoparticles of poly(D,L-lactic acid) (PLA) or poly(D,L-lactic-co-glycolic acid) (PLGA) covered by chemically esterified amphiphilic hyaluronate (HA) which will be used for intra-articular injection as a drug carrier for the treatment of arthritis (RA) and/or osteoarthritis (OA). PLA and PLGA are FDA approved polymers that are already used for the preparation of nano or microparticles. HA is a natural polysaccharide already present in the articulations known to interact with the CD44 receptors of the cells (especially chondrocytes). Therefore, we can envisage that the HA covering can improve the interactions between the cells and the nanoparticles, leading to better targeting or biodistribution. The knee of healthy male rats was injected one to two times weekly, with various concentrations of nanoparticles encapsulating Dextran-FITC. The synovial membranes and the patellae were collected aseptically and histologically analyzed to assess the effects and localization of the nanocapsules in the knee joint. We did not observe significant modifications in the synovial membranes (weak hyperplasia) or patellae integrity after local administration of nanodevices into the rats. While we found some nanoparticles in the synovial membrane, none were detected in the patellae. Moreover, the histological observations for patellae were confirmed by radiosulfate intake, which depicted no decrease in proteoglycans biosynthesis in nanoparticles treated animals. Concerning the safety towards synovial membranes, we also had a look at the inflammatory response after injections of nanoparticles covered by amphiphilic HA or polyvinyl alcohol (PVA) by monitoring the mRNA expression levels of some specific early cytokines (IL-1β and TNF-α). Once again, no differences were observed between the control rats and the rats treated with nanoparticles. Considering these preliminary results obtained in healthy rats, we can establish that neither the amphiphilic HA-covered PLGA nanoparticles nor their degradation products induce major modifications of articular tissues functions, while injected into the knee of healthy rats. These results should be confirmed in OA or RA rat models, in order to confirm that nanoparticles do not worsen already altered (degenerative or inflamed) articular tissues. Once confirmed, such tuneable nanoparticles could be proposed as a safe drug delivery system for the treatment of articular disease, allowing a wide range of encapsulating molecules.

Local induction of heat shock protein 70 (Hsp70) by proteasome inhibition confers chondroprotection during surgically induced osteoarthritis in the rat knee

AIM to determine if chondrocytic Hsp70 induction, via intra-articular injections of a reversible proteasome inhibitor (MG132), can protect articular chondrocytes from cellular death in experimental rat OA knee induced surgically by anterior cruciate ligament transection (ACLT). MATERIALS AND METHODS ACLT was performed on D0. Histological lesions in naive (sham) controls (ACLT+saline) and treated (ACLT+MG132) rats were assessed according to Mankins score. Repeated intra-articular injections (1.5 muM MG132 or saline were performed on D1, D7, D14 and D21. Rats were sacrificed sequentially on D7, D14 and D28. Detection of active caspase-3 and protein expression of Hsp70 was also determined on D7, D14 and D28 by immunostaining methods. RESULTS MG132 significantly reduced OA lesions on D28 in the MG132 treated group. The expression of Hsp70 increased 11-fold in the MG132-treated group versus 2-3-fold in ACLT-control rats on D28. Concomitantly, cells expressing caspase-3 increased 4-fold in ACLT model and decreased 2-fold with MG132 treatment. CONCLUSIONS Intra-articular induction of Hsp70 by MG132 could be a safe and interesting tool in chondrocytes protection from cellular injuries and thus might be a novel chondroprotective modality in rat OA.

New trends in MRI of cartilage: Advances and limitations in small animal studies

Due to the actual interest for bioengineering in the osteoarthritis (OA) healing context, researchers need accurate qualitative and quantitative methodologies to evaluate in vivo the integration and functionality of their cartilage-like biomaterials. As in clinical diagnostic strategies, advances in Magnetic Resonance Imaging (MRI) seem promising for non-vulnerant assessments of articular cartilage bio-architecture and morphology in small animal models. These experimental models are commonly used to monitor the physiopathology of OA and to evaluate therapeutic responses mediated by chondroprotective drugs or tissue engineering. Nowadays, the application of MR protocols to in vivo small animal cartilage imaging is achievable with the development of high magnetic fields and the adaptation of methodologies to reach the required spatial resolution and contrast. The purpose of this article is to summarize these current MRI strategies used for in vivo small animal articular cartilage assessments.

Osteogenic differentiation of human bone marrow mesenchymal stem cells in hydrogel containing nacre powder

Nacre (or mother of pearl) can facilitate bone cell differentiation and can speed up their mineralization. Here we report on the capability of nacre to induce differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) and the production of extracellular matrix. hBM-MSCs were encapsulated in an alginate hydrogel containing different concentrations of powdered nacre and cultured in the same environment until Day 28. Analysis of osteogenic gene expression, histochemistry, second harmonic generation (SHG) microscopy, and Raman scattering spectroscopy were used to characterize the synthesis of the extracellular matrix. In the presence of nacre powder, a significant increase in matrix synthesis from D21 in comparison with pure alginate was observed. Histochemistry revealed the formation of a new tissue composed of collagen fibers in the presence of nacre (immunostaining and SHG), and hydroxyapatite crystals (Raman) in the alginate beads. These results suggest that nacre is efficient in hBM-MSCs differentiation, extracellular matrix production and mineralization in alginate 3D biomaterials.

New trends in articular cartilage repair

Damage to the articular cartilage is an important, prevalent, and unsolved clinical issue for the orthopaedic surgeon. This review summarizes innovative basic research approaches that may improve the current understanding of cartilage repair processes and lead to novel therapeutic options. In this regard, new aspects of cartilage tissue engineering with a focus on the choice of the best-suited cell source are presented. The importance of non-destructive cartilage imaging is highlighted with the recent availability of adapted experimental tools such as Second Harmonic Generation (SHG) imaging. Novel insights into cartilage pathophysiology based on the involvement of the infrapatellar fat pad in osteoarthritis are also described. Also, recombinant adeno-associated viral vectors are discussed as clinically adapted, efficient tools for potential gene-based medicines in a variety of articular cartilage disorders. Taken as a whole, such advances in basic research in diverse fields of articular cartilage repair may lead to the development of improved therapies in the clinics for an improved, effective treatment of cartilage lesions in a close future.

Effect of dynamic loading on MSCs chondrogenic differentiation in 3-D alginate culture

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-β) treatment under the 3-dimensional (3-D) culture condition. In addition to these molecular and biochemical methods, the mechanical regulation of differentiation and matrix formation by MSCs is only starting to be considered. Recently, mechanical loading has been shown to induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of a calibrated agitation on the chondrogenesis of human bone MSCs (MSCs) in a 3-D alginate culture (day 28) and on the maintenance of chondrogenic phenotypes. Biomechanical stimulation of MSCs increased: (i) types 1 and 2 collagen formation; (ii) the expression of chondrogenic markers such as COMP and SOX9; and (iii) the capacity to maintain the chondrogenic phenotypes. Notably, these effects were shown without TGF-β treatment. These results suggest that a mechanical stimulation could be an efficient method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering in a 3-D environment. Additionally, it appears that MSCs and chondrocyte responses to mechanical stimulation are not identical.

Innovative TCSPC-SHG microscopy imaging to monitor matrix collagen neo-synthetized in bioscaffolds

We propose an innovative invasiveless technique in the field of nonlinear optical imaging to facilitate monitoring of cell/scaffold combinations for tissue repair. By using a near infrared (NIR) femtosecond excitation, we were able to introduce a new index based on decay time response for fluorescence (F) and Second Harmonic Generation (SHG) obtained with Time Correlated Single Photon Counting (TCSPC) microscopy to monitor structural information on the state of the matrix collagen. Some human Mesenchymal Stem Cells (hMSCs) seeded in 3D scaffolds were tested with different culture times (from D7 to D56) to analyze the effect of Tumor Growth Factor beta 1 (TGF-β1) on type-2 collagen expression in the matrix. After 14 days in the presence of TGF-β1, our results showed an increase in the expression of type-2 collagen synthesized by hMSCs, and a change in collagen conformation, as an indication of its ability to be detected as a harmonophore by TCSPC-SHG without the need for an exogenous probe.

Ambivalent properties of hyaluronate and hylan during post-traumatic OA in the rat knee.

AIM To determine whether viscosupplementation with intra-articular (i.a.) low- or high-molecular-weight hyaluronate (HA) injections influenced both chondral and synovial lesions in rats with surgically-induced OA knee. METHODS On D0, rats underwent anterior cruciate ligament transection (ACLX) and were divided in 4 groups: sham group, ACLX-saline control group, ACLX-hyaluronate group, ACLX-hylan group. IA injections were performed on D7, D14 and D21. Histological grading of chondral and synovial lesions were performed on D28. Concomitant immunostainings of Caspase3a and Hsp70 were also performed. RESULTS Articular damages were significantly reduced in both HAs-treated knee joints. In contrast, a significant increase of histological score of synovial inflammation was noted in both ACLX + HAs groups. Apoptotic events significantly decreased as anti-apoptotic Hsp70 expression increased significantly in both HAs groups. CONCLUSION HAs may exert, independently of its molecular weight, ambivalent properties on articular structures, simultaneously exerting chondroprotective properties and promoting long-term subacute synovitis.

Expression of chondrogenic genes by undifferentiated vs. differentiated human mesenchymal stem cells using array technology.

This study investigated the gene expression profile of human mesenchymal stem cells seeded in collagen sponge for 28 days in three different mediums: (1) basal medium as control containing ITS alone, (2) ITS+TGF-β1 alone or (3) ITS 1% supplemented sequentially by TGF-β1 (D3-D14) followed by BMP-2 (D15-D28). Differential expression of 84 genes implicated in chondrogenic and osteogenic differentiation of MSCs was analyzed at D28 by real-time RT-PCR array technology. TGF-β1 alone down-regulated two genes, CD36 and cathepsin K. Sixteen genes were significantly up-regulated, notably type 2 and type 10 collagens, COMP and Sox9. The sequential combination of TGF-β1 and BMP-2 produced a similar profile with prominent expression of type 2 collagen and the alkaline phosphatase gene. Interestingly, in this in vitro condition, RUNX2 was not up-regulated, suggesting that the sequential combination of TGF-β1/BMP2 enhances the hypertrophic chondrogenic profile without turning towards the osteoblastic pathway.

Hypoxia for MSC expansion and differentiation: the best way for enhancing TGFß-induced chondrogenesis and preventing calcifications in alginate beads

We examined the respective influence of a sequential or a continuous hypoxia during expansion and transforming growth factor beta 1-driven chondrogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The differentiation was performed within alginate beads, a classical tool for the implantation of MSCs within the joint. The standard normoxic 2D (expansion) and 3D (differentiation) MSCs cultures served as reference. To determine the quality of chondrogenesis, we analyzed typical markers such as type II and X collagens, SOX9, COMP, versican, and aggrecan mRNAs using polymerase chain reaction and we assessed the production of type II collagen and hypoxia-inducible factor (HIF)-1α by histological stainings. We simultaneously assessed the expression of osteogenic mRNAs (Alkaline Phosphatase, RUNX2, and Osteocalcin) and the presence of micro-calcifications by Alizarin red and Raman spectroscopy. Chondrogenic differentiation is clearly improved by hypoxia in 3D. Best results were obtained when the entire process, that is, 2D expansion and 3D differentiation, was performed under continuous 5% hypoxic condition. In addition, no calcification (hydroxyapatite, proved by RAMAN) was observed after 2D hypoxic expansion even in the case of a normoxic differentiation, in contrast with controls. Finally, a better chondrogenic differentiation of human MSCs is achieved when a reduced oxygen tension is applied during both expansion and differentiation times, avoiding in vitro osteogenic commitment of cells and subsequently the calcification deposition.