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

Comparison of TCP and TCP/HA Hybrid Scaffolds for Osteoconductive Activity

Two types of porous ceramic scaffolds were prepared, consisting of β-tricalcium phosphate (TCP) or the mixed powder of TCP and hydroxyapatite (HA) at a 2:1 mass ratio. A variety of methods have been used to fabricate bone scaffolds, while the sintering approach was adopted in this work. An extremely high temperature was used on sintering that proposed to consolidate the ceramic particles. As revealed by SEM, a well opened pore structure was developed within the scaffolds. The θ-values were measured to be of 73.3° and 6.5° for the composite scaffold and TCP sample, respectively. According to XRD patterns, the existence of grains coalescence and partial bonding between HA and TCP powders was demonstrated. Scaffold mechanical property in the term of flexural strength was also determined. The result showed decreasing of the strength by HA supplement, suggesting the more brittle characteristic of HA in comparison with TCP. By soaking the composite scaffold in PBS for a period of 2 weeks, transformation from particles to flank-like crystalline was clearly observed. Such change was found to be favorable for cell attachment, migration, and growth. By implanting cell-seeded scaffolds into nude mice, an abundant osseous extracellular matrix was identified for the composite implants. In contrast, the matrix was minimally detected in TCP implanted samples. Thus, the composite scaffold was found superior for hard tissue regeneration

Order versus Disorder: in vivo bone formation within osteoconductive scaffolds

In modern biomaterial design the generation of an environment mimicking some of the extracellular matrix features is envisaged to support molecular cross-talk between cells and scaffolds during tissue formation/remodeling. In bone substitutes chemical biomimesis has been particularly exploited; conversely, the relevance of pre-determined scaffold architecture for regenerated bone outputs is still unclear. Thus we aimed to demonstrate that a different organization of collagen fibers within newly formed bone under unloading conditions can be generated by differently architectured scaffolds. An ordered and confined geometry of hydroxyapatite foams concentrated collagen fibers within the pores, and triggered their self-assembly in a cholesteric-banded pattern, resulting in compact lamellar bone. Conversely, when progenitor cells were loaded onto nanofibrous collagen-based sponges, new collagen fibers were distributed in a nematic phase, resulting mostly in woven isotropic bone. Thus specific biomaterial design relevantly contributes to properly drive collagen fibers assembly to target bone regeneration

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

BRITER: A BMP Responsive Osteoblast Reporter Cell Line

BACKGROUND: BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. METHODOLOGY/PRINCIPAL FINDINGS: We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line), responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. CONCLUSION: As the dynamic range of the assay (for BMP responsiveness) is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance

Les cellules hypophysaires produisent-elles des ''Bone Morphogenetic Proteins'' (BMPs)?

National audienc

Identification of issues and environmental impacts for machining process by chip breaking conventional machines and control digital workshop tools in general factory Jose Maria Cordova

Los procesos de conformado por eliminación de material, es denominado proceso de mecanizado, se caracterizan por la obtención de una geometría final de una pieza mediante la eliminación del material sobrante. Según el método empleado en la eliminación de material, puede considerarse incluidas dentro de los procesos de mecanizado las siguientes categorías. Procesos convencionales en esta eliminación de material se realiza fundamentalmente por medios mecánicos. Los procesos de torneado(Se conoce como tornear al proceso de mecanizado realizado con un torno (máquina-herramienta para tornear) en el cual la pieza o barra gira y mediante herramientas acopladas se pueden realizar distintas operaciones de mecanizado, tales como, cilindrado sobre el eje o interiores, roscados, agujeros, ranuras o distintas formas.), fresado(Una fresadora es una máquina herramienta utilizada para realizar el movimiento de una herramienta rotativa de varios filos de corte denominada fresa. las piezas fresadas pueden ser desbastadas o afinadas.) y taladrado, pertenecen a este grupo. Procesos no convencionales en esta eliminación se hace mediante otros medios diferentes de los mecánicos , estos procesos son la electroerosión y fresado químico .Los procesos de mecanizado constituyen, en la actualidad, el conjunto de procesos de fabricación más ampliamente difundidos en la industria. Los mecanizados de precisión se realizan en torno CNC (control numérico), y las grandes series de piezas, se realizan en tornos automáticos, aunque aún quedan muchos mecanizados que se realizan en torno manual para la construcción de prototipos o piezas de con demasiada precisión.Forming processes by removing material, is known machining process, characterized by obtaining the final geometry of a workpiece by removing the excess material. According to the method employed in the removal of material, may be included within the machining processes of the following categories. Conventional processes with material removal is mainly accomplished by mechanical means. The process of turning (This is known as turning the machining process performed with a lathe (machine tool for turning) in which the part or rod rotates and through coupled tools can perform various machining operations such as turning operation on inner shaft or threaded holes, slots or different ways.), milling (a milling machine is a machine tool used for the movement of a rotary tool cutting edges called several strawberry. milled parts can be polished or honed.) and drilling, belong to this group. Processes in this unconventional removal is done by means other than mechanical, these processes are chemical milling and EDM machining processes are .The, currently, all the manufacturing processes more widespread in the industry. The precision machining is performed on CNC (numerical control), and the great series of pieces, performed in automatic lathes, although there are still many machining operations performed on manual lathe for building prototypes or parts too precisely

The performance of BMP-2 loaded TCP/HAP porous ceramics with a polyelectrolyte multilayer film coating

International audienceDelivering rhBMP-2 (recombinant Bone Morphogenic Protein-2) at low but therapeutically efficient dose is one of the current challenges for bone tissue repair. In this context, Polyelectrolyte Multilayer films (PEM) represent an attractive rhBMP-2 carrier due to their ability to protect proteins from denaturation and to coat a wide variety of materials with complex geometry. Herein, we coated macroporous TCP/HAP granules with a biopolymeric PEM film to deliver rhBMP-2 in a "matrix-bound" manner. In vitro release kinetics indicated that the PEM-coated granules sequestered significant amounts of rhBMP-2. The degree of film cross-linking influenced the quantity of rhBMP-2 trapped within the films. Bare (uncoated) TCP/HAP scaffolds were also able to retain rhBMP-2. Bioactivity of rhBMP-2 in the PEM-coated granules was confirmed on two cell markers: luciferase expression on BMP-responsive-element/Luc C2C12 cells and alkaline phosphatase activity induction on C2C12 cells. Promisingly, rhBMP-2 adsorbed onto PEM-coated and on bare granules in a lesser extent, could be stored and remained bioactive over at least 3 weeks. In vivo, both uncoated and PEM-coated TCP/HAP granules loaded with rhBMP-2 exhibited both osteoconductive and osteoinductive properties. This opens perspective for coating these bioactive PEM on other types of implantable materials, including metal alloy that do not exhibit any affinity for rhBMP-2. (C) 2011 Published by Elsevier Ltd

Desferrioxamine-driven upregulation of angiogenic factor expression by human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are the subject of intense research because of their biological properties and potential use for the repair of damaged tissues. Success of BMSC-based therapies, however, relies on a number of methodological improvements, including the establishment of a vascular network providing nutrients and oxygen to the transplanted cells and ensuring their immediate survival and long-term functionality. We described a method to enhance the autocrine expression of angiogenic factors by BMSCs. For this purpose, human BMSCs were treated with desferrioxamine (DFX). No PDGF-BB, VEGF-R1 or -R2 mRNA expression was detected under any of the conditions tested. mRNA and protein expression levels of TGFbeta1 were similar in BMSCs, whether they were exposed to DFX (50 microM) or to control conditions under normoxia for 48 h. In comparison with the results obtained with control conditions under normoxia, exposure of BMSCs to DFX for 48 h resulted in upregulation of bFGF at the protein (26-fold) but not at the mRNA levels and VEGF at both the mRNA (1.5-fold) and protein levels (4.5-fold). In comparison with the results obtained with control conditions under hypoxia, DFX induced a 50% increase in VEGF secretion but led to the same level of hypoxia inducible factor-1alpha protein expression (a transduction factor involved in angiogenic factor expression and known to be activated by DFX). Exposure of BMSCs to DFX resulted in oversecretion of angiogenic factors, suggesting that DFX-treated BMSCs could be used to supply angiogenic factors