In Vivo bone tissue induction by freeze-dried collagen-nanohydroxyapatite matrix loaded with BMP2/NS1 mRNAs lipopolyplexes

Messenger RNA (mRNA) activated matrices (RAMs) are interesting to orchestrate tissue and organ regeneration due to the in-situ and sustained production of functional proteins. However, the immunogenicity of in vitro transcribed mRNA and the paucity of proper in vivo mRNA delivery vector need to be overcome to exert the therapeutic potential of RAM. We developed a dual mRNAs system for in vitro osteogenesis by co-delivering NS1 mRNA with BMP2 mRNA to inhibit RNA sensors and enhance BMP-2 expression. Next, we evaluated a lipopolyplex (LPR) formulation platform for in vivo mRNA delivery and adapted the LPRs for RAM preparation. The LPR formulated BMP2/NS1 mRNAs were incorporated into an optimized collagen-nanohydroxyapatite scaffold and freeze-dried to prepare ready-to-use RAMs. The loaded BMP2/NS1 mRNAs lipopolyplexes maintained their spherical morphology in the RAM, thanks to the core-shell structure of LPR. The mRNAs release from RAMs lasted for 16 days resulting in an enhanced prolonged transgene expression period compared to direct cell transfection. Once subcutaneously implanted in mice, the BMP2/NS1 mRNAs LPRs containing RAMs (RAM-BMP2/NS1) induced significant new bone tissue than those without NS1 mRNA, eight weeks post implantation. Overall, our results demonstrate that the BMP2/NS1 dual mRNAs system is suitable for osteogenic engagement, and the freeze-dried RAM-BMP2/NS1 could be promising off-the-shelf products for clinical orthopedic practice.info:eu-repo/semantics/publishedVersio

Secondary structure of rhBMP-2 in a protective biopolymeric carrier material

Efficient delivery of growth factors is one of the great challenges of tissue engineering. Polyelectrolyte multilayer films (PEM) made of biopolymers have recently emerged as an interesting carrier for delivering recombinant human bone morphogenetic protein 2 (rhBMP-2 noted here BMP-2) to cells in a matrix-bound manner. We recently showed that PEM made of poly(l-lysine) and hyaluronan (PLL/HA) can retain high and tunable quantities of BMP-2 and can deliver it to cells to induce their differentiation in osteoblasts. Here, we investigate quantitatively by Fourier transform infrared spectroscopy (FTIR) the secondary structure of BMP-2 in solution as well as trapped in a biopolymeric thin film. We reveal that the major structural elements of BMP-2 in solution are intramolecular β-sheets and unordered structures as well as α-helices. Furthermore, we studied the secondary structure of rhBMP-2 trapped in hydrated films and in dry films since drying is an important step for future applications of these bioactive films onto orthopedic biomaterials. We demonstrate that the structural elements were preserved when BMP-2 was trapped in the biopolymeric film in hydrated conditions and, to a lesser extent, in dry state. Importantly, its bioactivity was maintained after drying of the film. Our results appear highly promising for future applications of these films as coatings of biomedical materials, to deliver bioactive proteins while preserving their bioactivity upon storage in dry state.This work was supported by the French Ministry of Research through an ANR-EmergenceBIO grant (ANR-09-EBIO-012-01), by the European Commission (FP7 program) via a European Research Council starting grant (BIOMIM, GA 259370), and by GRAVIT (081012_FIBIOS). C.P. is grafetul to IUF for financial support

Strategies for improving the efficacy of bioengineered bone constructs: a perspective

Bioengineered bone scaffolds are intended for use in large bone defects. Successful bone constructs should stimulate and support both the onset and the continuance of bone ingrowth. In an attempt to improve their performance and to compete with the one of autologous bone grafts, a growing symbiosis at the biological and material level is required. Recent advances have been made to further exploit the osteogenic potential of MSCs in scaffold development. Current research encompasses new strategies for reducing cell death after implantation and the manufacturing of tailored, instructive scaffolds.status: publishe

la tension d'oxygène régule les fonctions paracrines des cellules souches mésenchymateuses

International audienceMesenchymal stem cells (MSCs) have captured the attention and research endeavors of the scientific world because of their differentiation potential. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly due to the multitude of bioactive mediators secreted by these cells. Because the paracrine potential of MSCs is closely related to their microenvironment, the present study investigated and characterized select aspects of the human MSC (hMSC) secretome and assessed its in vitro and in vivo bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. In contrast to supernatant conditioned media (CM) obtained from hMSCs cultured at either 5% or 21% of O2, CM from hMSCs cultured under near anoxia exhibited significantly (p < .05) enhanced chemotactic and proangiogenic properties and a significant (p < .05) decrease in the inflammatory mediator content. An analysis of the hMSC secretome revealed a specific profile under near anoxia: hMSCs increase their paracrine expression of the angiogenic mediators vascular endothelial growth factor (VEGF)-A, VEGF-C, interleukin-8, RANTES, and monocyte chemoattractant protein 1 but significantly decrease expression of several inflammatory/immunomodulatory mediators. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine and could contribute to improving the efficacy of such therapies.SIGNIFICANCE:The present study investigated and characterized select aspects of the human mesenchymal stem cell (hMSC) secretome and assessed its in vitro and in vivo biological bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. The present study provided the first evidence of a shift of the hMSC cytokine signature induced by oxygen tension, particularly near anoxia (0.1% O2). Conditioned media obtained from hMSCs cultured under near anoxia exhibited significantly enhanced chemotactic and proangiogenic properties and a significant decrease in the inflammatory mediator content. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine, could contribute to improving the efficacy of such therapies, and most importantly highlighted the interest in using conditioned media in therapeutic modalities.Les cellules souches mésenchymateuses (CSM) sont très attractives pour la thérapie cellulaire en raison de leur potentiel de différenciation. Cependant, de nombreuses preuves suggèrent que les effets bénéfiques des CSM sont principalement dus à la multitude de médiateurs bioactifs sécrétés par ces cellules. Parce que le potentiel paracrine des CSM est étroitement lié à leur microenvironnement, la présente étude a examiné et caractérisé certains aspects du sécrétome du CSM humain (CSMh) et évalué sa bioactivité in vitro et in vivo en fonction de la tension en oxygène, en particulier près de l'anoxie. (0,1% O 2) et l’hypoxie (5% O 2), conditions qui reflètent l’environnement auquel les MSC sont exposés au cours de traitements in vivo à base de MSC. Contrairement aux milieux conditionnés (MC) obtenus à partir de CSM cultivées à 5% ou à 21% d'oxygène, les MC provenant de CSM cultivées presque en anoxie présentaient des propriétés chimiotactiques et proangiogéniques renforcées de manière significative (p <0,05) et une diminution significative du contenu en médiateur inflammatoire p<0,05). Une analyse du sécrétome des CSM a révélé un profil spécifique dans l'anoxie proche: les CSM accroissent leur expression paracrine du facteur de croissance endothélial vasculaire (VEGF)-A, VEGF-C, l'interleukine-8, RANTES et la protéine 1 de monocyte, un agent chimioattractant monocytaire mais diminue de façon significative l'expression de plusieurs médiateurs inflammatoires / immomodulateurs. Ces résultats fournissent de nouvelles preuves qui élucident des aspects d'une grande importance pour l'utilisation des CSM en médecine régénérative et pourraient contribuer à améliorer l'efficacité de tels traitements

Osteogenic-differentiated Mesenchymal Stem Cell-secreted Extracellular Matrix as a Bone Morphogenetic Protein-2 delivery system for ectopic bone formation

International audienceWhile human bone morphogenetic protein-2 (BMP-2) is a promising growth factor for bone regeneration, a major challenge in biomedical applications is finding an optimal carrier for its delivery at the site of injury. Because of their natural affinities for growth factors (including BMP-2) as well as their role in instructing cell function, cultured cell-derived extracellular matrices (ECM) are of special interest. We hereby hypothesized that a “bony matrix” containing mineralized, osteogenic ECM is a potential efficacious carrier of BMP-2 for promoting bone formation and, therefore, compared the efficacy of the decellularized ECM derived from osteogenic-differentiated human mesenchymal stem cells (hMSCs) to the one obtained from ECM from undifferentiated hMSCs. Our results provided evidence that both ECMs can bind BMP-2 and promote bone formation when implanted ectopically in mice. The osteoinductive potential of BMP-2, however, was greater when loaded within an osteogenic MSC-derived ECM; this outcome was correlated with higher sequestration capacity of BMP-2 over time in vivo. Interestingly, although the BMP-2 mainly bound onto the mineral crystals contained within the osteogenic MSC derived-ECM, these mineral components were not involved in the observed higher osteoinductivity, suggesting that the organic components were the critical components for the matrix efficacy as BMP-2 carrier

Establishment of an in vivo model for molecular assessment of titanium implant osseointegration in compromised bone

Shortening of the healing time before loading risks impeding successful titanium implant anchorage into compromised bone. A thorough understanding at the genetic scale of the early phases of bone regeneration at the implant interface is required before the development of strategies to enhance implant osseointegration. In this study a new in vivo implant model to explore the mechanism by which titanium implant osseointegration is affected by the host bone properties is presented. An implant was conceptualized enabling standardized harvesting of peri-implant tissue for quantitative molecular analysis while preserving the mimicking of the clinical setting. The implant is partly indented to provide a well-defined healing compartment from where tissue differentiation and de novo bone formation can be investigated and partly screw-threaded to provide a good implant anchorage into the bone. The feasibility of the implant design was assessed in osteopenic bone conditions, evoked by simulated weightlessness. Wistar rats were either hindlimb unloaded by tail suspension (HU) for 9 days or acted as controls (CTL). The status of compromised bone tissue through 9-days HU was confirmed by micro-X-ray computed tomography. The implant was installed in the proximal tibial bone 7 days after the onset of HU or CTL. Two days postimplantation, the peri-implant regenerating tissue responses were recorded by measuring expression of inflammatory, angiogenic, and bone resorption parameters (hypoxia-inducible factor 1, alpha subunit; vascular endothelial growth factor A; angiopoietin 1; endothelial PAS domain protein 1; fibroblast growth factor 2; tumor necrosis factor; interleukin 11; acid phosphatase 5, tartrate resistant; tumor necrosis factor (ligand) superfamily, member 11/RANKL). We successfully demonstrated that HU-associated bone conditions evoked a significant alteration of expression of the angiogenic markers in the peri-implant regenerative tissue during initial implant osseointegration, whereas the expression levels of the inflammatory and bone resorption parameters remained unchanged. We concluded that this in vivo implant model provides a well-designed and controlled method to examine molecular responses in implant osseointegration to impaired bone conditions. This model may serve to explore the application of anabolic strategies in peri-implant osteogenesis.status: publishe

Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation

International audienceApplication of messenger RNA (mRNA) for bone regeneration is a promising alternative to DNA, recombinant proteins and peptides. However, exogenous in vitro transcribed mRNA (IVT mRNA) triggers innate immune response resulting in mRNA degradation and translation inhibition. Inspired by the ability of viral immune evasion proteins to inhibit host cell responses against viral RNA, we applied non-structural protein-1 (NS1) from Influenza A virus (A/Texas/36/1991) as an IVT mRNA enhancer. We evidenced a dose-dependent blocking of RNA sensors by NS1 expression. The co-delivery of NS1 mRNA with mRNA of reporter genes significantly increased the translation efficiency. Interestingly, unlike the use of nucleosides modification, NS1-mediated mRNA translation enhancement does not dependent to cell type. Dual delivery of NS1 mRNA and BMP-2 mRNA to murine pluripotent stem cells (C3H10T1/2), promoted osteogenic differentiation evidenced by enhanced expression of osteoblastic markers (e.g. alkaline phosphatase, type I collagen, osteopontin, and osteocalcin), and extracellular mineralization. Overall, these results support the adjuvant potentiality of NS1 for mRNA-based regenerative therapies

Bone Regeneration in Sheep Using Acropora Coral, a Natural Resorbable Scaffold, and Autologous Mesenchymal Stem Cells

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In vivo molecular evidence of delayed titanium implant osseointegration in compromised bone

Optimization of implant osseointegration in patients with reduced bone healing potential is a challenge remaining in implant dentistry. Identification of the genes that are modulated during implant osseointegration in normal versus osteopenic bone is needed to successfully address these pertinent clinical needs. The present study aimed to assess the initial and early molecular events following titanium implant installation in normal and compromised bone in a rat tibia model. Peri-implant tissue from a well-defined tissue regeneration compartment was analyzed at 2 and 7 days post-surgery for the expression of select markers of inflammation, angiogenesis, bone resorption and bone formation. Impaired bone was induced by hindlimb unloading and validated using μCT. The essential step of angiogenesis preceding bone regeneration was evidenced for the peri-implant setting in healthy bone. Compromised bone significantly affected the angiogenesis-osteogenesis coupling in the initial phase (2 days post-surgery), with altered expressions of Vegfa and Epas1 coinciding with downregulated expressions of Col1a1, Bmp2, Bmp4, Alpl and Bglap. At 7 days post-implantation, differences between normal and compromised peri-implant bone were no longer observed. This in vivo molecular evidence of delayed implant osseointegration in compromised bone reassert modern strategies in implant development, such as surface modifications and bioengineered approaches, to improve implant osseointegration in compromised conditions.status: publishe

Comparison of Survival and Osteogenic Ability of Human Mesenchymal Stem Cells in Orthotopic and Ectopic Sites in Mice

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