FR2917426A1 - REACTOR FOR IMPLEMENTING A BONE TISSUE CULTURE PROCESS - Google Patents

Abstract

The invention relates to a method for culturing bone tissue. Said method being of the type in which: particles of a material compatible with the bone tissue are provided; bone cell stem cells are provided; said particles are seeded with said bone cell stem cells; providing a nutritional fluid (31) containing nutritional elements for proliferating bone cells; said nutritional elements are delivered to said bone cell stem cells through a flow of said nutritional fluid (31); and according to the invention said bone cell stem cells are maintained within said flow such that said flow exerts mechanical stresses on said cells and said bone cells proliferate on said particles (35) to form large tissue implantable bone. These granules of implantable bone tissue are then recovered for transplanting.

Description

Reactor for carrying out a bone tissue culture process

  The present invention relates to a method and a device for culturing bone tissue. The bone graft is widely practiced, for example when following the removal of tumors or infected bone tissue, it is necessary to fill this loss of bone tissue, sometimes important, by bone transplantation. Also, an autologous bone graft is transplanted to fill the bone space left free by the excision. Therefore, it is first necessary to determine on the patient by imaging of the affected bone part, the morphological parameters of the bone space to be filled, its shape and dimensions. Then, the graft is taken from another healthy bone of said patient taking into account the morphological parameters mentioned above. Then, the graft is implanted in a massive form in said space to be filled so as to obtain a mechanical continuity in said bone part reached. However, the removal of a graft from the patient already weakened by his pathology is not always desirable or possible. Also, it was imagined to artificially reproduce bone grafts to the desired size and then transplant them into the bone space to be filled. In particular, it will be possible to refer to US 2005/002910, in which a reactor is described which makes it possible to produce large bone grafts, for example the head of a femur. However, not only is the time required for the production of such bone grafts relatively long, but moreover it must be consistent with the bone space to be resorbed, which requires relatively complex additional means, and in particular deformable reactor walls. .

  Also, a problem that arises and that aims to solve this

  invention is to provide a device for implementing a method of

  bone tissue culture, which allows rapid tissue production

  bone ready to be implanted and also relatively simple to implement. In order to solve this problem, according to a first aspect, the present invention provides a method for culturing bone tissue, said method being of the type in which particles of a material compatible with bone tissue and stem cells are provided. of cells

  bone; then said particles are seeded with said bone cell stem cells; also, a nutritional fluid containing nutritional elements for proliferating bone cells is provided and said nutritional elements are delivered to said stem cells of bone cells via a flow

  Said nutritional fluid; according to the invention, said bone cell stem cells are maintained within said flow such that said flow exerts mechanical stresses on said cells and said bone cells proliferate on said particles to form implantable bone tissue granules; and finally, we recover

  20 said implantable bone tissue granules for transplanting.

  Thus, a feature of the invention resides in the mode of production of bone tissue by proliferating mesenchymal stem cells on particles independent of each other through the flow of a nutritional fluid that not only allows

  25 supply of nutritional elements, but also exerts mechanical stresses on the cells, which further stimulates their proliferation. Advantageously, said flow of nutritional fluid to the wall of said particles forming a parietal boundary layer forms a flow capable of exerting parietal shear stresses.

  Between 104 and 10.2 Pa in said parietal boundary layer. These parietal shear stresses are for example of the order of 10-3 Pa. In this way, individualized bone tissue granules are obtained which can then be transplanted into the bone space to be filled. Thus, to a certain extent, there is no need to predict in advance, or even to measure beforehand the volume of the bone space to be filled, since the granules of bone tissue are produced in relatively large numbers, and they are then inserted per pack into the bone space until it is completely filled. Then, these granules of bone tissue transform very quickly into a solid bone implant that extends into the bone space to be filled.

  According to a preferred embodiment of the invention, there is provided

  advantageously a mineral material for providing said particles, for example tricalcium phosphate or calcium carbonate, whose density is much higher than the density of water. The advantage of this density will be explained in greater detail in the remainder of the description. A natural osteoconductive coral-based ceramic constitutes a

  It is a preferred material for forming these particles because it is particularly inexpensive. In addition, it is relatively easy to produce particles calibrated in this material.

  Preferably, porous particles are provided to allow the cells to proliferate within these particles, as

  This will be explained in the detailed description, which allows faster reconstitution of healthy bone tissue after transplantation. And the aforementioned coral-based material has the advantage of being itself porous.

  Advantageously, resorbable particles are provided, that is to say particles whose material disappears little by little after the implantation of the

  25 graft to disappear completely eventually. The above-mentioned coral-based particles have this quality. In a preferred manner, particles having a size of between 0.8 and 5 mm are provided, and for example particles of substantially cubic shape with sides of 3 mm are provided. The aforementioned mineral material is easily

  Packaged in cubic particles of this size.

  According to another preferred embodiment of the invention, the pH of the nutritional fluid which flows between the seeded particles and in which the cells are immersed, is maintained at a value of between 7 and 8, for example 7.4 . In another aspect, the present invention provides a bone tissue culture reactor, said reactor comprising a longitudinal culture chamber in which particles of a bone-compatible material are maintained, said particles being intended to be seeded with bone tissue. bone cell stem cells, said reactor comprising means for delivering nutritional elements to the seeded particles via

  a flow of nutritional fluid; the reactor comprises means for maintaining said bone cell stem cells within said flow such that said flow exerts mechanical stresses on said cells and said bone cells proliferate on said particles to form tissue granules

  Implantable bone. Thus, the reactor according to the invention makes it possible to maintain the bone cells at the heart of the flow of nutritional fluid, which not only enables the nutritional elements to be delivered to the cells so that they proliferate, but also to exert constraints mechanical

  20 on these cells that promote their proliferation.

  According to a particularly advantageous embodiment of the reactor, said culture chamber is a cylindrical tube with a circular director, having a dimer D and a length L, the ratio of length L and diameter D being between 10 and 15. This tube

  For example, cylindrical cylinders are made of a plastic material, which is ideally transparent at an advantageous cost, so that it can be thrown away after a culture cycle, for example 21 days. The particles are introduced and placed in the reactor so as to occupy a circular cylindrical space of a height h and a diameter d, the ratio of the

  The height h and the diameter d being preferably between 4 and 6, for example 5, so as to obtain optimum flow characteristics around the particles.

  Advantageously, said culture chamber is oriented vertically and said particles are held in said culture chamber resting on a retaining grid, so that the nutritional fluid flows upwardly through said grid and into the culture chamber. Thus, all the particles, in the culture chamber, undergo a flow to their walls, substantially identical. In addition, said means for delivering nutritional elements to said seeded particles comprise pumping means

  for pumping a nutritional fluid into a reservoir at a predefined rate to produce said flow of nutritional fluid in the culture chamber.

  Advantageously, said pumping means make it possible to create a flow whose flow rate is between 8 and 15 ml / min.

  15 to create a substantially laminar flow to the wall of said seeded particles without causing them to suspend in the nutritional fluid. Thus, during the flow, the particles remain in abutment on each other, retained by said grid.

  Other features and advantages of the invention will emerge from the

  Reading the following description of a particular embodiment of the invention, given by way of indication but not limitation, with reference to the accompanying drawings in which:

  - Figure 1 is a schematic view of a reactor for carrying out a bone tissue culture method according to the invention;

  Figure 2A is a schematic view of a portion of a reactor as illustrated in Figure 1; and,

  Figure 2B is a numbering diagram relating to the reactor portion shown in Figure 2A.

  Figure 1 illustrates a reactor for bone tissue culture

  According to the invention. The reactor comprises a cylindrical tube 10 of plastic material with a circular director, with an outside diameter of about 33 mm, and a total height H of 215 mm. The inside diameter of the cylindrical tube 10 is however between 10 and 15 mm, for example 12 mm. The cylindrical tube 10 defines a culture chamber 11 with a height close to 160 mm. Note that the drawings of Figure 1 are not reproduced in the proportions of the dimensions given above for the sake of clarity.

  A filling chamber 12 is formed at the base of the cylindrical tube 10 and has a height of 40 mm for a diameter of 26 mm. A support grid 14 is mounted between the culture chamber 11 and the filling chamber 12, and extends along a straight section of the

  This support grid 14 is formed of a disk pierced with orifices 15 1 mm in diameter, which orifices are spaced apart by a distance of 2 mm.

  The cylindrical tube 10 is mounted substantially vertically on a base 16 to seal it tightly, and this base

  15 16 has an inlet opening 18 which opens into the filling chamber 12 opposite the support grid 14. This inlet opening 18 is connected to a reservoir 20 with a capacity of at least 250 ml, via an inlet conduit 22, which inlet conduit 22 passes through a peristaltic pump 24. In addition, the inlet opening 18

  20 is extended by a T-tube 26 located inside the filling chamber 12 and near the inlet opening 18; the T-tube 26 having two opposite openings oriented transversely in a direction substantially perpendicular to the cylindrical tube 10.

  In addition, the reactor has a shutter 28 allowing

  Sealing the cylindrical tube 10 in its upper part. And the shutter 28 has an outlet opening 30 connected to the reservoir 20 via a return duct 32.

  Thus, the peristaltic pump 24 is adapted to suck in the reservoir 20 a nutritional fluid 31 which will be described hereinafter and for the

  30 to flow in the filling chamber 12 so that it can flow into the culture chamber 11 through the support grid 14, and then escape through the outlet opening 30 to return, in a closed circuit in the reservoir 20 through the return conduit 32. In this way, the nutritional fluid flows upwardly in the culture chamber 11. It will be noted that the closed circuit of nutritional fluid is preferably sealed against to the outside atmosphere.

  Moreover, thanks to the T-tube 26, the nutritional fluid flows into the filling chamber 12 and through the orifices 15 of the support grid 14 according to uniform flow characteristics for the entire surface of the grid. In reality, the characteristics of this flow which, upstream of the culture chamber 11, are dependent on the pump

  Are the peristaltic 24, and in particular the instantaneous velocity of the flow, damped by the T-tube 26. Thus, is the upward flow of the nutritional fluid inside the culture chamber 11 homogeneous along a section of FIG. this room of culture 11.

  The bone tissue culture method according to the invention makes

  Also called particles of a mineral material, and in this case particles based on coral Pontes species. This material consists essentially of calcium carbonate and the particles formed in this material are essentially of cubic geometry with sides of about 3 mm in length. Moreover, this material is

  And is also absorbable as will be explained hereinafter.

  These particles will then be loaded into the culture chamber 11 bearing on the support grid 14, but before they are seeded with bone cell stem cells. These stem cells, mesenchymal stem cells (MSCs) and

  The species a murine C3H1OT1 / 2 cell line containing 106 cells per milliliter and, expressing a fluorescent marker, GFP, easily detectable by fluorescence microscopy, are sown on the aforementioned particles.

  The seeded, free particles are then loaded into the culture chamber 11 and above the support grid 14 over a height of between 40 and 60 mm. The seeded and charged particles form between them interstices through which the nutritional fluid will be able to flow.

  With regard specifically to the nutritional fluid, incorporating nutritional elements and in particular BME (Based Medium Eagle) and fetal calf serum (FCS), it is introduced into the reservoir 20 and injected into the filling chamber 12 at a rate of 10. ml per minute. In this way, the filling chamber 12 is gradually filled with nutritional fluid, which then flows through the support grid 14 and between the seeded particles of the culture chamber 11.

  10 ml per minute is sufficiently moderate, and the particles are sufficiently dense relative to the density of the nutritional fluid, so that the flow does not cause the separation of the seeded particles. These seeded particles remain in packed bed during the flow, that is to say in support on each other and they

  15 are retained by the support grid 14. It is then assumed as a first approximation, that the porosity of all the particles sown in packed bed, which is equivalent to the volume of the space occupied by the particles minus the total volume of the particles , divided by the said volume of the space they occupy, is close to 0.5.

  Thus, after having loaded 250 particles of the aforementioned type, previously inoculated with a total of 236 million stem cells, after 15 days of culture, for a flow of nutrient fluid of 10 ml per minute, 1,500 million cells, more than six times more. The nutritional fluid of the reservoir 20 is, however, replaced every

  Three days, because it is depleted in nutritional elements because of the proliferation of cells and also it contains cellular waste.

  Furthermore, the surface of a cell on which the nutritional fluid exerts stresses is substantially equivalent to 10.'0 m2. The cells adhering to the particles, they are then subjected to

  30 shear stresses of viscous origin from the nutritional fluid, which then appear within the parietal boundary layer or the boundary layer to the wall. These are the stresses that exert forces and forces on these cells. Given the size of the bioreactor and a flow rate of nutrient fluid of 10 ml per minute, we obtain a Reynolds number characteristic of the flow, close to 10. This number is characteristic of a Poiseuille flow. Viscosity-dominated, it is also well below 2000, the value of the Reynolds number from which the flow is no longer laminar and becomes turbulent. Moreover, considering that all the packed-bed particles constitute a porous medium through which the nutritional fluid flows, it is then possible to define equivalent channels or pores through which the fluid flows. The Reynolds number characteristic of the flow in these channels is then substantially divided by two,

  It is close to 5. A fortiori, the flow of the nutritional fluid through these channels or pores is characteristic of laminar flows.

  The parietal shear stresses in these channels are then, by applying the conventional laws of the flow of fluids in a tube, of approximately 4.10-3 Pa. Also, the associated forces referred to the

  The surface area of a cell is of the order of 4.10-13 N, ie 40 pico Newton (pN). Granules of a bone biohybrid material consisting of a coral particle coated with bone cell type cell colonies are then obtained. We will first refer to Figure

  2A, schematically showing strata 33 of coral particles 35 on top of each other and first 34, in contact with a support grid 14 'at the fourteenth and last, 36, located towards the 30 'exit aperture, and then to Figure 2B illustrating the amount of cells present on the granules of the different strata

  30 after 21 days of culture. This quantity of cells is counted by the cell proliferation index, which is the ratio of the number of cells at the instant t = 21 days and the number of cells initially seeded.

  The index and cell proliferation is plotted on the abscissa on a scale from 0 to 10, while the number of each of the strata is plotted on the y-axis on a scale from 1 to 14 and corresponding precisely to the successive strata. Thus, in the form of a histogram, the diagram of Figure 2B, reports horizontally for each of the strata 33, the proliferation index in the form of a thick line whose end corresponds to this index.

  Thus, it is observed that the proliferation of the cells varies between 7 and 8 times the number of initial cells after 21 days of culture and this, what

  the stratum of particles considered between the first 34 and the fourteenth 36. Therefore, it appears that the proliferation of the cells is substantially identical on all the strata of seeded particles, from the first 34 to the fourteenth 36, which means that the conditions of contribution in nutritional elements are substantially

  Identical, regardless of the position of the particles within the culture chamber 11. This means that the flow conditions are relatively homogeneous around the seeded particles, from the first 34 to the last stratum 36.

  This characteristic is decisive since the material

  A biohybrid ready to be implanted and having a plurality of granules must have a relatively homogeneous cell concentration for all the granules. It will be noted that all the bone cells cultured for 21 days on 250 particles according to the embodiment described above, make it possible to fill a bone defect of a

  Volume between 850 and 900 mm3. However, if it is desired to fill a larger bone space, it is possible to a certain extent to increase the number of particles seeded initially loaded in the culture chamber 11. If the bone space to be filled is very important, or if multiple bone spaces need to be filled

  At the same time, it is necessary to provide a reactor of a larger size, the dimensions of which would be determined by homothety with respect to the aforementioned reactor. For example, the dimensions of such a new reactor could be twice that shown in Figure 1. By means of an adjustment of the flow rate of nutritional fluid, it would then obtain flow conditions of this nutritional fluid, between the particles seeded, totally similar to those obtained in the reactor described here. In addition, according to the embodiment of the invention illustrated in FIG. 1, the absence of abrasion of the granules of the biohybrid material is verified throughout the culture, which abrasion could come from the flow of the fluid. nutritional. For this, the calcium level of the nutritional fluid is measured during the 21 days of culture and it is verified that it oscillates between 3 and 4 millimoles per liter. Moreover, cells that multiply on coral particles do not come off. Indeed, if we initially measure a quantity of one million cells in the nutritional fluid, after six days, there remains only about 250,000 and 15 this number does not evolve until the 21st day. In addition, during the 21 days of culture, the pH of the nutritional fluid remains constant at around 7.4. It should be noted that the maintenance of the pH at a physiological pH is crucial for the survival and development of the mesenchymal stem cells; and that contrary to prior art techniques, the method according to the invention does not require any particular regulation of this pH. In addition, the particles of the material used, based on coral Porites species, constitute porous matrices within which the bone cells can also proliferate.

Thus, thanks to the bone tissue culture method according to the invention and to the reactor allowing its implementation, the proliferation of bone cells from mesenchymal stem cells is optimal because the transfer of nutrients, oxygen and cell waste in particular, between the nutritional fluid and the cells 30 operate quickly in view of the flow of the nutritional fluid through the particles. Diffusion and convective material transfers are by nature increased relative to cells cultured in a static nutritional fluid. In addition to the material transfers, which feed the cells, the mechanical stresses, in particular pressure and shear, exerted on the cells by the nutritional fluid which flows between the particles, make it possible to increase the proliferation and the differentiation of the cells. stem cells. Thus, the culture method according to the invention makes it possible to increase very significantly the productivity of the bone cell culture. In addition, the reactor implementing the method operates in a closed circuit and only the reservoir of nutritional fluid is changed during a production cycle, which avoids not only contaminations of the bone cell culture, but also the contact time. manipulation. Moreover, the tank change can be automated.

Moreover, the design of the reactor is relatively simple and it can be made of a disposable material at an advantageous cost.

Claims (16)

  A method of culturing bone tissue, said method being of the type in which: particles (35) of a material compatible with bone tissue are provided; - Stem cells of bone cells are provided; said particles are seeded with said bone cell stem cells; providing a nutritional fluid (31) containing nutritional elements for proliferating bone cells; said nutritional elements are delivered to said stem cells of bone cells via a flow of said nutritional fluid (31); Characterized by maintaining said bone cell stem cells within said flow such that said flow exerts mechanical stresses on said cells and said bone cells proliferate on said particles (35) to form tissue granules implantable bone; And in that said implantable bone tissue granules are recovered for transplanting.   2. A culture method according to claim 1, characterized in that, said flow of nutritional fluid to the wall of said particles forming a parietal boundary layer, forms a flow capable of exerting wall shear stresses of between 10-4 and 10-2 Pa in said parietal boundary layer.   3. Culture method according to claim 1 or 2, characterized in that provides a mineral material to provide said particles (35).   4. Culture method according to any one of claims 1 to 3, characterized in that it provides calibrated particles.   5. Culture method according to any one of claims 1 to 4, characterized in that provides porous particles.   6. Culture method according to any one of claims 1 to 5, characterized in that it provides resorbable particles.   7. The culture method according to any one of claims 1 to 6, characterized in that said particles (35) have dimensions between 0.8 and 5 millimeters.   8. Culture method according to any one of claims 1 to 7, characterized in that provides particles (35) having a substantially cubic shape.   9. The culture method according to any one of claims 1 to 8, characterized in that said particles (35) comprise calcium carbonate.   10. The culture method according to any one of claims 1 to 9, characterized in that maintaining the pH of said nutritional fluid (31) between 7 and 8.   11. Reactor for bone tissue culture, said reactor comprising a longitudinal growth chamber (11) in which particles (35) of bone-compatible material are held, said particles being intended to be seeded with stem cells bone cells, said reactor comprising means for delivering nutrients to the seeded particles via a nutritional fluid flow (31); characterized by comprising means (10, 12, 14, 16, 26) for maintaining said bone cell stem cells within said flow such that said flow exerts mechanical stresses on said cells and said cells bones proliferate on said particles to form implantable bone tissue granules.   12. Reactor according to claim 11, characterized in that said culture chamber (11) is a cylindrical tube with a circular director, having a dimer D and a length L, the ratio of length L and diameter D being between 10 and 15.   13. Reactor according to claim 11 or 12, characterized in that said culture chamber is oriented vertically and in that said particles (35) are held in said culture chamber (11) resting on a retaining grid (14). .   14. Reactor according to claim 13, characterized in that said flow of nutritional fluid is produced through said grid (14).   15. Reactor according to any one of claims 11 to 14, characterized in that said means for delivering nutritional elements to said seeded particles comprises pumping means (24) for producing said nutrient fluid flow (31).   16. Reactor according to claim 15, characterized in that said pumping means (24) can create a flow whose flow rate is between 8 and 15 ml / min.