Today, I am going to present you the subject of my internship which is: the regeneration of the bone! As you suspect it, I did not present you the domains of biomaterials and tissular engineering previously by chance. Indeed, while combining these two domains we let us can regenerate bones. First of all, the laboratory in which I work is the LMGP (Laboratoire des Matériaux et du Génie Physique) within the team IMBM (Interfaces between Materials and Biological Matter). This laboratory is situated inside the scientific pole of Grenoble (see Fig 1.). Figure 1: the laboratory LMGP and the scientific pole of Grenoble [1] I am going to begin by explaining to you briefly how we work on the regeneration of the bone and then I would explain you more in detail the used techniques. My subject in some lines Then how it is possible to regenerate bones? It is possible with the regenerative medicine! In fact for it, we are going to combine techniques connected in biomaterials and to the tissular engineering. Indeed, the principle consists in forming films of polymers about implants which we load then with a protein which is a growth factor. It is going to allow the cells which are going to adhere on the implant to be in touch with the growth factor which is going to lead the differentiation of cells in osteoblasts that is more simply osseous cells. Here is a short video in French during a conference TED of my supervisor of internship explaining with vulgarisation our project [2]: The formation of the films of polymers To start, a polymer is a very long chemical molecule formed by the repetition of a motive which we call monomer. The films of polymers which we realize are called PEM (Polyelectrolyte Multilayer films). The polymers which we use are thus polyelectrolytes, they have a charge. We use poly-L-Lysine (PLL) as polycation (charged +) and the polyanion (charged -) is the Hyaluronic Acid (HA) (see Fig 2.). It is thanks to the interaction between these charges that we are going to be able to form our films of polymers. The PLL and the HA are bought under forms of powders then are dissolved in a buffer solution in a precise pH. Figure 2: Chemical formula of poly-L-Lysine (PLL) [3] and Hyaluronic Acid (HA) [4] The technique which we use to form our films of polyelectrolytes is called LbL for Layers By Layers technique deposition (see Fig 3.). As his name indicates it, we form films by forming successive layers of polymers. The films which we realize are films of PLL/HA. We put a layer of polycation which is the PLL then the layer of polyanion which is the HA and so on to form our films. In every stage of deposition of a layer, we realize rinsings so that films can form correctly. We work with films of 12 or 24 bilayers. We can make films on glasses slides or directly on implants by using various techniques as the dipping which consists in dipping samples successively into tubs containing the various solutions or still by using a spray to apply the solutions. Every technique possesses advantages and inconveniences which play a role on the properties of the film, the cost or the precision. We can also realize various stages of treatment of the film after its formation to modify its properties as for example by realizing a chemical treatment to change its rigidity. We analyze our films in the confocal microscope by marking our film with various fluorescent molecules. So, we can measure the thickness of our film or still quantify its homogeneity. Films can be formed on various types of implants. We can made it on implants in titanium, ceramic or still polymer. Figure 3: Formation of PEM with the LbL technique deposition [5] Bone Morphogenetic protein 2 (BMP-2) Bones Morphogenetics Proteins are proteins and more exactly growth factors. There are about ten approximately and play in particular an important role in the formation of several types of tissues in the body as the cardiac, muscular, neuronal or still osseous tissues (see Fig4.). For our project, we use the BMP-2 which allows the cells to differ in osteoblasts. Having formed our film of PLL/HA correctly about a glass slide or an implant, we incorporate the BMP-2 into our film. Figure 4: 3D structure of BMP-2 [6,7]
Finally, the last stage consists in putting cells in cultures on our film for the in vitro studies or otherwise on the implant within a body for the in vivo studies. We can observe the adhesion of our cells with differents types of microscopies as by marking the actin of cells then by analyzing the images in the confocal. We can also verify if our cells differed well by using various tests as enzymatic tests. The majority of the tests which we make are in vitro with cells C2C12 which are cells of mouse which can differ in muscles or in bone. We also use stem cells of mouse. We also already made in vivo tests on rabbits and rats which were a success. We managed to treat fractures in a few weeks while it is normally very difficult. In summary My internship concerns the regenerative medicine and more exactly the regeneration of bones. For that purpose, we form films of polyelectrolytes which we load with a growth factor which allows the cells to differ in osseous cells. We have already obtained from in vitro many interesting results and the first in vivo results are very encouraging. It is thanks to the interdisciplinarity from the team and from the project that we managed to obtain these results by combining the domains of biomaterials and tissular engineering. Thanks to these researches, we have new perspectives for the regenerative medicine because we hope to be able using this technique with other growth factors to be able to regenerate other tissues in the body and also treating cancerous tissues. Thank you for your reading and see you soon! References: [1] http://www.lmgp.grenoble-inp.fr/le-laboratoire/?RH=LMGP_Pr%C3%A9sentation [2] https://www.youtube.com/watch?v=gk7X5gbcz_w&feature=youtu.be [3] http://www.sigmaaldrich.com/catalog/product/sigma/p9155?lang=fr®ion=FR [4] http://www.sigmaaldrich.com/catalog/product/sigma/49775?lang=fr®ion=FR&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1 [5] http://eng.thesaurus.rusnano.com/wiki/article1563 [6] https://en.wikipedia.org/wiki/Bone_morphogenetic_protein [7] http://www.rcsb.org/pdb/explore.do?structureId=1REU
5 Comments
Nicolas
22/11/2016 02:36:58 pm
Hi Loïc !
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Loïc
23/11/2016 12:34:04 pm
Hi Nicolas,
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Nicolas
23/11/2016 12:49:52 pm
Hi Loïc !
Loïc
11/12/2016 11:01:33 am
Hi Nicolas!
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Nicolas
12/12/2016 06:43:04 am
Hi Loïc,
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