Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
  • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/20—Material Coatings
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/14—Scaffolds; Matrices
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/10—Perfusion
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
  • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
  • A61C8/0003—Not used, see subgroups
  • A61C8/0004—Consolidating natural teeth
  • A61C8/0006—Periodontal tissue or bone regeneration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
  • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
  • A61C8/0087—Means for sterile storage or manipulation of dental implants

Definitions

• Bioreactor for tissue cultured in thin layer uses
• the present invention relates to a cell and tissue culture reactor, optionally for the preparation of an implant, in particular by cell growth and / or differentiation, optionally in a biocompatible matrix and / or on an adhesion support.
• the bioreactor is particularly suitable for the culture of tissue or cells in thin layer (s), and comprises means making it possible to ensure physiological stimulation of the tissues or cells and a supply in culture medium, adapted to the culture in thin layer.
• the reactor also advantageously comprises means or a form making it possible to facilitate the introduction or the extraction of the tissue or of the solid part of an implant into the culture chamber, and for example a wall or a deformable element. elastically.
• the reactor may also include means for regulating the temperature.
• the reactor can be used for the cultivation of different cell types and the preparation of implants of various shapes, compositions and applications.
• the present invention also relates to methods for preparing implants using a bio-reactor according to the invention.
• the invention can therefore be used in particular for the preparation of dental, bone, cartilaginous implants, etc., comprising a tissue or a cellular composition, optionally associated with a biocompatible matrix and / or with a solid or semi-solid adhesion support. cells or tissues or matrix.
• a reactor for manufacturing dental implants is known in particular from document WO 00/21456.
• This reactor makes it possible to receive an implant core comprising a root part and a coronary part.
• the core consists of an inert biocompatible material in a shape analogous to an extracted tooth.
• the root part of the implant is immersed in a culture of mesenchymal cells, in a culture medium whose composition allows differentiation into cementoblasts and fibroblasts. This immersion is maintained for a time sufficient to allow adhesion of the cementoblasts on the root part, the formation of a first layer of cement and a blank of dental alveolus ligament attached to this cement.
• the reactor is designed to allow mechanical stress on the tissue being formed around the implant. This stress reproduces the physiological constraints to which a tooth is normally subjected. Indeed, physiological stimulation is necessary to allow proliferation and differentiation in vitro of cementoblasts and fibroblasts.
• the core of the implant is placed in a cell delimited by a rigid porous wall through which a culture medium is diffused.
• mechanical means are arranged between the core of the implant and the reactor vessel. These means ensure a back and forth movement of the core of the implant relative to the vessel. The displacement takes place in particular along the length of the implant.
• the excitation means comprise for example an electric actuator.
• Such a reactor is difficult to manufacture since it must allow precise guiding in translation of the core of the implant relative to the vessel. Indeed, the space between the wall of the cell and the nucleus must be maintained within a determined range of values for satisfactory growth of the cementoblasts and fibroblasts. In addition, this reactor does not allow renewal or control of the composition of the medium surrounding the cells. On the other hand, the mechanical stimulation offered has certain limits. In addition, this reactor is described specifically for the preparation of a dental implant.
• the object of the invention is to provide reactors which are easier to manufacture and which allow satisfactory growth and / or differentiation of the cells in the reactor chamber, preferably within a matrix (eg, extracellular matrix produced by cells or cultured tissue) and on the surface of a support (adhesion), more particularly of cell or tissue cultured in a thin layer.
• a matrix eg, extracellular matrix produced by cells or cultured tissue
• a support e.g., cell or tissue cultured in a thin layer.
• This reactor is in particular to allow the control of several culture conditions to promote the proliferation and cell differentiation, extracellular matrix production, tissue organization and maturation.
• the bioreactor is more particularly suitable for the culture of tissues or cells in a thin layer, in particular of structural tissue, for which there are numerous constraints and difficulties.
• the first condition is a surface effect which allows cell adhesion or tissue attachment to a biocompatible wall in the reactor or to two opposite walls, separated by the culture space which is then subjected to a double effect. surface or attachment.
• the second condition is a mechanical effect generated within the tissue to bring it closer to its natural physiological conditions.
• the stresses can be in pressure, in stretching, in shearing, in friction or in a combination of these effects.
• Another constraint resides in the supply of culture medium, allowing good homogeneous growth of the tissue in a thin layer, that is to say a homogeneous and controlled supply of the medium in the reduced space of the culture.
• the present invention thus provides for the first time a biological culture reactor, suitable for the culture of tissue in a thin layer, ensuring mechanical stimulation of the cells and a suitable supply and diffusion of the nutritional medium.
• the reactor is also provided with temperature control means and means allowing or facilitating the introduction or the extraction of the solid part of an implant (such as the adhesion support) in the culture space. without altering the cells or tissues present.
• An object of the invention therefore resides, more precisely, in a reactor for cell or tissue culture in thin layer (s).
• the reactor is more particularly characterized in that it comprises (i) a culture chamber delimiting, between two walls, a culture space of between 1 and 1000 microns, preferably between 10 and 1000 microns, more preferably between 50 and 800 microns, (ii) means for mechanically stimulating cells or tissues in said space and (iii), preferably, means for perfusing the culture medium within the culture space.
• a culture chamber delimiting, between two walls, a culture space of between 1 and 1000 microns, preferably between 10 and 1000 microns, more preferably between 50 and 800 microns
• means for mechanically stimulating cells or tissues in said space preferably, means for perfusing the culture medium within the culture space.
• the bio-reactor according to the invention can make it possible to obtain, for a therapeutic, scientific or industrial purpose, biological tissues as such.
• This bio-reactor also makes it possible to cultivate and develop tissues on the surface of an adhesion support or of a prosthesis which will keep on their surface or on a part of their surface the cultivated tissue, for example to improve or accelerate the tissue healing and organic repair after surgical placement of the mixed implant or said prosthesis thus produced.
• the invention can be implemented with different types of implants, of varied shape, structure and composition and for different applications.
• the term implant in fact designates any composition comprising cells or a reconstituted tissue, possibly associated with a solid support. It is preferably an implant comprising a solid nucleus (or component) and a cellular or biological component.
• the implant core is also referred to herein as the "adhesion support" or heart of the implant.
• the implant core can be made of any biocompatible material, for example metal, plastic, polymer, glass, biological material, ceramic, bone, coral, etc. Typically, it is bioverre, ceramic (for example zirconia), alloys, titanium or bone.
• the implant according to a specific example of the invention is a dental implant, comprising a nucleus (root part) partially covered with cells by means of a reactor according to the invention.
• a dental implant for example, can be made of any material biocompatible, for example metal (titanium for example), polymer, biological glass, ceramic, etc. It can also be other types of implants, such as for example a bone or joint implant.
• the bioreactor according to the invention advantageously comprises a culture chamber delimiting, between two walls, a culture space suitable for the production of tissue in thin layer (s). It is advantageously a culture space with a thickness less than or equal to about 1000 microns, generally between about 10 and about 1000 microns, more preferably between 50 and 800 microns. Preferably, the culture space has at least one zone of thickness less than or equal to 900 microns, more preferably around 800 microns, preferably between 50 and 800 microns.
• the reactor is suitable for the formation of biological tissues of controlled and low thickness, for example of structural tissues, for example of the sheet type.
• the culture space is advantageously homogeneous within all or part of the culture chamber, so as to ensure the production of an essentially homogeneous cultured tissue.
• the thickness of the culture space is advantageously constant in a large area of the chamber.
• the walls delimiting the culture space can be made up or include different types of materials, rigid or not.
• the walls delimiting the culture space are made of biocompatible material, for example polymer, glass, plastic, metal, etc. More preferably, at least one of the walls is made of a material promoting cell proliferation, or cell adhesion, or reproducing a physiological situation.
• particularly preferred materials are biogreen, bone, coral, hydroxyapatite, titanium oxide, etc. (see also examples).
• the reactor comprises a culture space delimited by two walls of biocompatible material, at least one of them being made up or base of a material favoring the culture of cells, preferably in bioverre, bone, coral.
• the material promoting cell culture can be in partial or total covering of other biocompatible materials, such as ceramics, metals or polymers.
• the two walls can be fixed or mobile, as will be described later.
• at least one of the two walls is movable.
• one of the walls can be formed by the core of the implant itself.
• the reactor chamber is delimited by a wall of shape adapted to that of the implant core, the introduction of which into the chamber creates the reduced culture space.
• the reactor chamber is delimited by a wall of conical or cylindrical shape and the core of the implant is respectively of conical or cylindrical shape.
• the reactor further comprises means for supporting the core of the implant, arranged or adjustable so that the introduction of the core of the implant forms, with the wall of the culture chamber, a culture space. reduced as defined above.
• the reactor culture chamber is delimited by a non-rigid wall, elastically deformable, making it possible to facilitate the introduction or the withdrawal of the implant core from the chamber, without damaging the tissue.
• the bioreactor further comprises means for introducing or removing the nucleus from an implant in the culture chamber.
• the invention also aims to provide a reactor making it possible to improve the conditions of introduction or extraction of the adhesion support (or core) of an implant in the culture chamber or cell, in particular without damaging the tissues adherent to the surface of the nucleus.
• the reactor is characterized in particular by the presence of a membrane delimiting or bordering a cell (or a chamber) for culture, differentiation and / or cell growth, said membrane being elastically deformable to facilitate introduction cells or that of the adhesion support in said chamber.
• the deformable membrane can also allow mechanical stimulation of the cells in said chamber.
• the present invention proposes and relates to a reactor for the preparation of an implant, in particular by cell growth and / or differentiation, optionally within a biocompatible matrix, comprising a cell for the culture, the differentiation and / or cell growth, characterized in that the alveolus is delimited by an elastically deformable membrane.
• the invention is based in part on the development of a chamber with a deformable wall, intended to receive the cells and, where appropriate, the adhesion support (or core) of an implant.
• the use of such a cell provides multiple advantages, and in particular that of facilitating the introduction of the cells or of the support of the implant into said chamber, and / or of allowing mechanical stimulation of the cells in said chamber.
• the reactor makes it possible, under sterile and reproducible conditions, to culture, differentiate and / or grow the cells on the surface of the implant nucleus .
• an adhesion support solid part composed for example of bone, polymer, biocompatible material, bioverre, teflon, metal, etc.
• the reactor makes it possible, under sterile and reproducible conditions, to culture, differentiate and / or grow the cells on the surface of the implant nucleus .
• the cells in gel or in suspension or in exogenous biological matrix or in self-secreting matrix, both in the tank and on the nucleus of the implant or the prosthesis.
• the cells can be identical or different in the tank and on the nucleus of the implant or prosthesis.
• the deformability properties of the wall in particular its elastic characteristics, make it possible to exert, during culture, mechanical stimulation on the cells, and to promote their proliferation and / or differentiation.
• the present invention therefore also relates to any bioreactor comprising a culture chamber delimited by an elastically deformable wall.
• the tank of the bio-reactor thus comprises, in an intermediate chamber, an elastic wall deformable by depression.
• the reactor according to the invention comprises means for mechanical deformation of the wall, in particular pneumatic or hydraulic means.
• the reactor according to the invention advantageously comprises means for mechanical stimulation of the cells or tissues cultured.
• the culture space can be subjected to constraints by relative movements of the two walls, the biological or micro-mechanical tissue attachment to one or to the two opposite walls producing mechanical stimulation.
• the mechanical effect is preferably exerted in pressure, in stretching, in shearing, in friction or in a combination (s) of these effects. It is generally obtained by movement of one or both walls of the chamber, essentially producing a change in the thickness of the culture space.
• the modification of the thickness is produced by movement of only one of the two walls, and is controlled not to exceed + or - approximately 20% of the initial thickness of the culture space, preferably + or - 10%.
• the reactor comprises means for mechanical stimulation of the cells, for example by means of a piston, by displacement of the nucleus of the implant, if necessary, or by deformation of the culture chamber or of a part of it (for example by means of a deformable seal).
• a displacement this can for example be exerted along the axis of the implant, by a movement back and forth, or by a slight partial rotation.
• the stimulation is carried out by means of a deformable element (for example, flexible), introduced into the reactor (membrane of the cell, joint, etc.) and by application of pressure / depression or a crushing force.
• elements to the culture space, such as absorbable or non-absorbable sponges, for example collagen, chitosan or collagen-chitosan sponge. , or else surface irregularities of one of the walls, for example grooves, or else fibers for example of biocompatible polymer, or balls with a diameter of 50 to 1000 microns, preferably from 50 to 500 microns, slightly less than the thickness of the culture space, and which may be made of polymer, glass, ceramic, etc., or a combination of these elements.
• absorbable or non-absorbable sponges for example collagen, chitosan or collagen-chitosan sponge.
• surface irregularities of one of the walls for example grooves
• fibers for example of biocompatible polymer
• balls with a diameter of 50 to 1000 microns, preferably from 50 to 500 microns, slightly less than the thickness of the culture space, and which may be made of polymer, glass, ceramic, etc., or a combination of these elements.
• a preferred embodiment of the invention relates to a bioreactor as defined above comprising (i) a culture chamber delimiting, between two walls, a culture space with a thickness less than about 1000 microns, (ii) means for mechanically stimulating cells or tissues in said space and (iii), preferably, means for perfusing the culture medium within the culture space, the culture space further comprising elements making it possible to increase the mechanical stimulation (sponge, fiber, ball, particle, etc.) or having at least one wall having irregularities (grooves, bends, fibers, etc.).
• a bioreactor as defined above, comprising (i) a culture chamber delimiting, between two walls, a culture space with a thickness less than about 1000 microns, (ii) means for mechanically stimulating cells or tissues in said space and (iii), preferably, means for perfusing the culture medium within the culture space, at least one of the two walls being made of material promoting culture cellular, preferably in bioverre or bone.
• the invention more generally resides in a bioreactor comprising a culture chamber, said chamber being delimited by an internal wall produced by or comprising bioverre or bone.
• the reactors of the invention also comprise means for perfusing the culture medium within the culture space.
• Another advantageous characteristic of the bioreactors of the invention is to promote the diffusion of the nutritional medium in a culture space reduced in thickness, necessary for obtaining a surface and interface effect in the cultivated tissue. The diffusion of the medium is obtained by perfusion but its distribution is obtained by a periodic expansion-contraction of this space and / or a movement of amplitude as regular as possible on all surfaces.
• the effect then approximates the blood pulse, and can be adjusted in amplitude and frequency for sufficient and suitable nutrition and functional stimulation, without risking destroying the tissue.
• the frequency of stimulation in particular expansion-contraction cycles is advantageously between 1 and 80 cycles per minute.
• the angle between the axis of movement of the rigid parts constituting the interface and the culture surface is an important element for obtaining a good adjustment.
• the pressure variations applied in the intermediate chamber make it possible to communicate the expansion-contraction effect which will promote the diffusion of the nutritive medium and the functional stimulation, or part of this stimulation, to the cultivated tissue.
• the culture space can vary by 10% around an average value of 50 to 1000 microns depending on the cultures.
• the number of inputs and outputs from the culture medium can be adapted to the tissue to be cultivated and to the material of the cell.
• the input can also come from the implant core, for example.
• a rigid cell in addition to or as a replacement for the previous solution, multiple inputs and outputs are possible to optimize the distribution of the medium in the culture space ( Figures 5 and 6).
• the inlets and outlets are arranged in plan, at 90 ° from one another, the outlets and inlets being alternated to obtain optimum diffusion of the medium. Channels dug on the surface in the wall of the cell can also help the diffusion of the medium.
• the reactor therefore also comprises a perfusion system, making it possible to supply and / or renew the culture medium present in the cell.
• the infusion rate can be adjusted by a person skilled in the art depending on the cell type and the type of culture, medium, etc.
• the diffusion of the medium within the reduced culture space is advantageously favored by the movement of one or more walls of the culture chamber (pressure, elastic deformation, back-and-forth movement, etc.) .
• the reactors of the invention also comprise means for regulating the temperature of the culture medium.
• Said means are for example a water or liquid or gas circuit, of controlled temperature, the presence of an electrical resistance, or of a transistor, for example of a Peltier effect transistor.
• the invention also relates to any cell or tissue culture reactor, characterized in that it comprises a culture chamber and means for regulating the temperature of the culture chamber. It is advantageously a reactor as defined above, comprising means of mechanical stimulation of the cells, and / or means of perfusion of the medium.
• the reactors according to the invention can be used for the culture of different cell types and the preparation of implants of various shapes, compositions and applications.
• it is used for the preparation of cellular products comprising cells whose growth, culture, differentiation and / or adhesion is favored by mechanical stimulation.
• Mention may in particular be made of fibroblasts, cementoblasts, chondrocytes, etc.
• Particular examples of cells are fibroblasts of the dermis, of the oral mucosa, of the gum, of alveolo-dental ligaments (desmodontals), of chondrocytes, or of precursors of these cells.
• the cell populations used can be mixed populations, comprising different cell types.
• the cells used in the invention can be autologous, allogenic or xenogenic. They can be primary cultures or established lines. They are preferably human cells or cells of human origin. They can be used in the form of a suspension, aggregates, clusters, carpets, possibly in a natural or synthetic extracellular matrix making it possible to facilitate adhesion to the nucleus of the implant.
• the cells can be genetically modified cells, that is to say cells containing a recombinant nucleic acid enabling them to be given properties of interest.
• the present invention also relates to the use of a bioreactor as defined above for the preparation of a tissue or a cell culture or an implant.
• the present invention also relates to a process for the preparation of an implant, cultured tissue or cell culture, by means of a reactor as defined above.
• the composition or cell tissue (which may include several types of cells) can be placed in the bio-reactor tank before it is closed. It can also be placed in adhesion to the surface of the adhesion support of the implant or prosthesis before they are placed in the bio-reactor tank.
• These two techniques can be used simultaneously, by placing cells in gel or in suspension or in an exogenous biological matrix or in a self-secreting matrix, both in the tank and on the adhesion support of the implant or the prosthesis. In this case, the cells can be identical or different in the tank and on the implant or prosthesis.
• the bio-reactor is adapted to provide a relatively thin culture space around the implant. Two situations can occur depending on the shape of the implant or prosthesis.
• the part can be placed or removed from the bio-reactor, without tearing off or scratching by friction the tissues adhering to its surface, due to a so-called "draft" shape with respect to an axis of insertion and establishment or withdrawal.
• the surfaces intended for cultivation can for example have a conical shape.
• the implant core or the prosthesis has a shape capable of causing friction on the cultivated surfaces, during installation in the bio-reactor or removal of the part from the bio-reactor, it is advantageous to '' use a deformable socket as described above, which can be spaced from the implant at these times.
• the subject of the invention is a reactor as defined above, for the preparation of an implant comprising a core covered with a matrix composed in whole or in part of cells, said reactor comprising:
• a specific example of implementation of the invention consists of a reactor for the preparation of a dental implant by cell growth on a root part of the implant, comprising:
• the membrane is elastically deformable and the mechanical stimulation means comprise means for deformation of said membrane.
• the invention relates to a reactor as defined above, for the preparation of an implant comprising a core covered with a matrix composed in whole or in part of cells, said reactor comprising:
• - a flexible or rigid wall delimiting or bordering a cell (or culture chamber) intended to receive all or part of the core of the implant; - means for supporting said implant core in the socket;
• the core support means further comprise an elastically deformable element (for example a seal), making it possible to ensure movement of the implant in the socket.
• an elastically deformable element for example a seal
• the cell is delimited by a rigid wall composed of or based on biogreen, bone or other biocompatible materials (polymer, glass, plastic, metal, or coral).
• the bioreactor further comprises means for regulating its temperature.
• the subject of the invention is a method of manufacturing an implant by growth and / or culture and / or cell differentiation on a solid adhesion support (eg, implant nucleus) within a medium. for culture, said method comprising: - bringing the nucleus of the implant into contact with cells, under conditions allowing adhesion of cells to said nucleus of the implant or to a part thereof;
• the method comprises: - bringing the nucleus of the implant into contact with cells, under conditions allowing adhesion of cells to said nucleus of the implant or to a part thereof;
• Another subject of the invention relates to a method for manufacturing an implant by growth and / or culture and / or cell differentiation on a solid adhesion support (eg, implant nucleus) within a culture, said process comprising:
• the term “adhesion” designates the fact that the cells (possibly in a matrix) can be maintained in contact with the nucleus of the implant, at least partially and temporarily, the time to introduce the nucleus into the cell or the reactor of the invention.
• This adhesion can be achieved by using surfaces naturally ensuring such adhesion or pre-treated for this purpose. It is also possible to include the cells in a gel, paste, sponge, self-secreting matrix, such as to facilitate this adhesion. It is also possible to cover the cells, after their deposition on the implant nucleus, with a film ensuring their maintenance, preferably with a porous biodegradable film.
• a particular example of application of the invention lies in the preparation of a dental implant, by culture of cells on the root part of said implant, introduced into a deformable socket according to the invention.
• the subject of the invention is also a method of cell culture, differentiation and / or proliferation, said method, comprising:
• the tissue is preferably perfused, so as to renew the culture medium.
• the perfusion is preferably provided by the presence of an opening in the lower part of the cell, ensuring the supply in fresh medium.
• This supply can also be carried out by a pipe provided in the nucleus (or body) of the implant, or in a plunger used for mechanical stimulation, if applicable.
• the outlet from the medium is generally provided by an opening located in the upper part of the cell.
• the medium used for cell culture can be any medium known to a person skilled in the art, in particular any medium suitable for the culture, growth or differentiation of mammalian cells. It may in particular be DMEM, RPMI, HAF, etc. medium, optionally supplemented with antibiotics, amino acids, serum, etc.
• the invention also relates to the use of a reactor as described above, or of a cell as described above, for the preparation of cellular compositions, in particular of implants, typically for use in the man.
• FIG. 1 is a view in longitudinal section of a reactor according to l the invention comprising a deformable wall
• Figure 2 is a top view of the reactor body according to the invention
• Figure 3 is a longitudinal sectional view of the reactor body according to the invention
• Figure 4 is a longitudinal sectional view of a transport container for a dental implant
• FIG. 5 is a view in longitudinal section of a reactor according to the invention, perfused for the culture of chondroblasts, comprising a rigid wall, with functional stimulation
• FIG. 6 is a view in longitudinal section of a reactor according to the invention, perfused for the culture of fibroblasts of the alveolodental ligament, with functional stimulation.
• the reactor 2 represented in FIG. 1 is intended in particular for the manufacture of a dental implant by cell growth on a nucleus of implant 4 made of a bio-compatible material.
• This core consists for example of ceramic, titanium, bio-glass or a composition of these materials.
• the reactor 2 is generally of revolution of axis x-x '. It comprises a tank 6 delimiting an enclosure 8.
• the tank 6 has a cylindrical body 10 and a cover 12 secured to the body.
• the body and the cover are both advantageously made of PTFE. It is understood that other materials can be used for the production of the reactor.
• the cover 12 is secured to the body 10 by three screws, not shown. The screws are engaged in passages 14 formed through the cover 12. The threaded end of the screws is received in tapped holes 16 formed in the body 10.
• blind holes 18, visible in FIG. 2 are provided in the facing faces of the cover and of the body to receive centering pins not shown.
• the enclosure 8 comprises an axial well 22 formed in the body 16. This well opens out at the center of the generally flat bottom 23 of a coaxial bowl 24 hollowed out in the body 10. This bowl opens onto a first plane end face 10A of the cylindrical body 10.
• An elastic membrane 26 in the shape of a bell is disposed in the enclosure 8. This membrane delimits a generally cylindrical cell 28 for receiving the root part, denoted 30, of the implant.
• the membrane 26 has a cylindrical section 32 closed by a bottom 34 pierced axially with a passage 36. At its end opposite the bottom, the membrane has an external flange 38 for securing the membrane 26 to the body 10. This flange came from material with the cylindrical section 32. It has on its face facing the bottom 34, a peripheral bead 40 received in a peripheral groove formed in the bottom 23 of the bowl.
• the membrane 26 is impermeable to liquids and gases and is deformable. It is formed for example from biocompatible silicone.
• the internal diameter of the cylindrical section 32 that is to say the diameter of the cell 28 is very slightly greater than the diameter of the root portion 30 of the implant.
• the difference in diameters is such that when the membrane 26 is at rest and that it is not deformed, the distance between the implant core 4 received in the socket and the membrane 26 is between 0, 1 and 5 mm.
• the annular space formed between the nucleus and the membrane corresponds to the tissue culture region.
• annular chamber 42 of non-zero thickness is formed between the body 10 and the membrane 26.
• the thickness of the annular chamber 42 is at least 1 mm. It is for example 3 mm.
• An axial bore 44 extends the well 22. This bore opens onto the second flat end face, denoted 10B, of the body.
• the bore 44 is normally closed by a pin 46 mounted tightly in this bore in order to close it sealingly.
• One end of the pin 46 projects inside the well 22. This end has a protuberance 47 adapted to engage elastically inside a recess 48 of complementary shape formed in the thickness of the bottom 34 of the membrane .
• the pin 46 has an internal conduit 49 opening axially at the top of the protuberance 47 opposite the passage 36 formed in the bottom 34 of the membrane.
• the conduit 49 opens, at the other end, from the pin 46, outside the reactor.
• the conduit 49 allows the supply of culture medium in the cell, by means of any suitable device (pump, syringe, etc.).
• the conduit 49 may also have a bend, so as to open onto the side wall of the reactor.
• a conduit 50 connected to the annular chamber 42 is formed through the body 10. This conduit is adapted to connect the chamber 42 to a source of variable vacuum, denoted 52.
• This source of variable vacuum consists for example of a pump empty.
• the duct 50 extends radially and opens into the side wall of the cylindrical body 10 where it has a connection profile to the vacuum source 52.
• an evacuation conduit 54 is formed through the body 10 for the circulation of a culture medium in which the root part 30 of the implant is immersed. This conduit opens, at one end, into the dish 24 and, at its other end, through the cylindrical wall of the body 10. This conduit is adapted to be connected to a collector 56 of culture medium.
• the reactor 2 comprises means 58 for supporting the core 4 of the implant in a fixed position relative to the vessel 6 and to the enclosure 8. These support means are engaged through a central orifice
• the support means 58 comprise a support pin 62 shown alone in FIG. 3.
• This pin 62 is generally of revolution. It comprises an shouldered axial passage 64 in which a rod, noted 66, is received, extending axially the core 4 of the implant.
• a through threaded hole 68 is formed radially through the wall of the pin 62.
• This hole receives a grub screw 70 for retaining the implant relative to the pin.
• the end of the screw 70 bears against a flat portion of the rod 66, thus ensuring its axial immobilization and in rotation.
• the rod 66 extends beyond the pin 62 at its end opposite to the implant core 4.
• This end of the rod is linked to the axial stud 72.
• This stud comprises, axially at one end, a housing in which the end rod 66 is received. The latter is held there by gluing.
• the stud 72 has an external thread 76 on which a nut 78 is attached.
• the pin 62 is received in a chamber 80 delimited by a cylindrical ferrule 82.
• the ferrule delimits a cylindrical passage opening at its two ends and in the length of which the chamber 80 is defined.
• the diameter of the passage is reduced by a re-entrant peripheral lip 84 forming a diaphragm.
• This re-entrant lip defines an annular bearing surface for the pin 62.
• the ferrule 82 has a thread adapted to receive a threaded closure plug 90 holding the pin 62 pressed against the lip 84.
• the pad 90 is axially traversed by a bore 92 for the passage of the stud 72. It has an external thread capable of cooperating with the tapping. Beyond the thread, the pad 90 has a knurled crown 94.
• the ferrule 82 has at its end having the lip 84 an outer collar 96 adapted to be clamped between the body 10 and the cover 12. This collar has a diameter greater than diameter of the bowl 24. Its outer peripheral edge is partially received in an annular cavity 100 formed in the first main face 10A of the body around the bowl 24. This cavity 100 has a flat bottom in which is formed an annular channel 102 receiving a O-ring seal 104.
• the depth of the cavity 100 is less than the thickness of the collar 96 so that the collar 96 and the ferrule 82 are kept tight between the bottom of the cavity 100 and the cover 12.
• the reactor is first assembled as illustrated in FIG. 1 while the core of the implant is not yet installed.
• the membrane 26 is placed in the well 22, and the ferrule 82 is engaged in the orifice 60 of the cover.
• the cover being immobilized on the body 10, the ferrule 82 is retained by the collar 96 sandwiched between the body and the cover.
• the core 4 of the implant is secured to the support pin 62 by the screw 70 retaining the rod 66 extending the core.
• the root part of the nucleus is incubated in the presence of a cell composition under conditions ensuring adhesion of cells to said root part.
• the cell composition typically comprises fibroblasts of the alveolo-dental ligament and / or precursors of these fibroblasts and cementoblasts.
• the root part of the nucleus is covered with cellular tissue. This cell tissue is previously cultivated flat and then wrapped around the root part of the implant nucleus.
• the root part of the nucleus is covered with a matrix impregnated with cells, for example of the sponge, gel, paste, etc. type.
• a depression is established in the annular chamber 42. This depression is created by the pump 52. Under the action of this depression, the membrane 26 is deformed in particular by radial expansion of its cylindrical section 32.
• the cell 28 is thus dilated increasing its internal section.
• the implant core is then introduced into the reactor 2 through the passage of the cover.
• the root part of the core is received in the socket 28.
• the pad 90 is engaged around the stud 72 and is screwed into the ferrule 82.
• the screwing is carried out until the support pin 62 is clamped and immobilized axially between the lip 84 and the tampon 90.
• the socket 28 is released by stopping the depression created in the chamber 42. It is understood that the invention is not limited to this mode of use, and that it is possible to bring the implant and the cell composition into contact directly in the cell of the reactor.
• the cell 28 For the culture of the cellular tissue, the cell 28 is permanently perfused with a culture medium introduced through the bottom 34 of the membrane by the conduit 49. The culture medium then circulates in the cell 28 along the nucleus 4 and comes out in the upper part of the cell to open into the bowl 24. The culture liquid is discharged from the bowl 24 through the discharge conduit 54.
• each cycle comprises a first phase tending to increase the volume of the cell 28 by establishing a depression in the sealed chamber 42 and a second phase during which the sealed chamber 42 is vented making the depression stop and tending to reduce this volume when the membrane relaxes by elasticity.
• This cyclic vacuum is caused by the vacuum source 52 connected to the chamber 42 by the conduit 50.
• the deformation of the membrane causes a mechanical action on the cells in culture.
• the pulsations created by the membrane in the culture medium favor the diffusion of the latter into the cell 28.
• the depression created by the source is periodic and its frequency can approach the natural pulse, without this reference being considered necessary for the result. For a human being, this frequency is such that 40 to 80 pulsations take place every minute. In the bioreactor, one pulse every three to ten seconds may be sufficient.
• the periodic depressions created in chamber 42 are advantageously repeated throughout the duration of the cell culture. They cause physiological stimulation of cells, capable of promoting tissue development.
• the chamber 42 delimited between the membrane 26 and the body 10 is compartmentalized by separation walls. Each closed compartment thus created is connected to its own vacuum source.
• the sources of vacuum are controlled successively to ensure successive deformations of the membrane and thus create a wave propagating along the membrane 26. This wave generates a pulsatile phenomenon in the culture space formed between the membrane and the nucleus of implant.
• the culture medium is perfused at several points distributed horizontally around the circumference of the tank.
• the inlets and outlets of the culture medium are located on either side of the implant core at multiple points on a vertical plane but diametrically opposite with respect to the implant core.
• the inputs and outputs are advantageously arranged along vertical lines, but at 90 ° from each other in plan and alternated to obtain optimal diffusion of the medium.
• the inputs and outputs can be inverted periodically so that all the cells are irrigated equally.
• the angle between the axis of movement of the rigid parts constituting the interface and the culture surface is an important element for obtaining a good adjustment.
• the pressure variations applied in the intermediate chamber make it possible to communicate the effect of dilation-contraction which will favor the diffusion of the nutritive medium and the functional stimulation, or part of this stimulation, to the cultivated tissue.
• the growing space can vary from
• the number of inputs and outputs of the culture medium can also be adapted to the tissue to be cultivated and to the material of the cell.
• an entry from the bottom of the tank and an exit from the top is the simplest method.
• multiple inputs and outputs are possible to optimize the distribution of the medium in the culture space. Channels dug on the surface in the wall of the cell can also help the diffusion of the medium.
• the nucleus 4 covered with the cultured cells is extracted from the reactor through the cover 12.
• the alveolus 28 is kept dilated by a depression created in the chamber 42.
• the implant is kept in a cylindrical container 130 illustrated in FIG. 4.
• This container has a cylindrical cavity 132 suitable for receiving the root part of the implant covered with cells.
• the cylindrical cavity 132 has a thread 134 allowing the threaded pad 90 to be screwed.
• the cavity has a shoulder 136 forming a bearing surface for the support pin 62.
• the support pin is immobilized axially by being clamped between the shoulder 136 and buffer 90.
• the distance between the bottom of the cavity and the shoulder 136 is such that when the support pin 62 is in abutment against the shoulder 136, the root part of the implant is at all points spaced from the wall of the cavity.
• the cavity is filled with a conservation liquid in which bathes the root part of the implant.
• the reactor shown in FIG. 5 comprises a tank 202 which preferably is made of bio-glass with a truncated-conical concavity to provide the interface with the internal cone 203 and thus obtain mechanical stimulation of the gel culture with an effect pressure and shear-friction which approaches the physiological conditions to which the cartilages are subjected.
• the size of the device can be adapted to the desired culture surface (frusto-conical), and therefore to the quantity of cells.
• the internal truncated cone 203 may also preferably be made of bioverre or any other material which mimics the support bone as much as possible. The flatter the cone, the greater the pressure variations and the diffusion of the medium, and the reduced shear stresses, and vice versa.
• the tank 202 can be inserted into a tank 201 provided with a cover 204 and having a flat bottom so as to allow the possible use of the reactor on a heating table and thus ensuring a homogeneous distribution of the temperature.
• the temperature regulation system can also be directly included in the wall of the tank 201 of the bio-reactor. It can, for example, be a water circuit or an electrical resistance or a Peltier effect transistor, etc.
• Two centering pins 213 are inserted in the cover 204 and the body 201.
• an inlet 212 intended to allow the penetration of the compressed air responsible for the movement and which thus allows the mechanical stimulation.
• a lower O-ring 210 allows the amplitude of the vertical movement applied to the middle. It is also possible to add an upper O-ring 211 to the device. which in turn facilitates the amplitude of the vertical movement applied to the pressurized air.
• the device further comprises a cylindrical movable part 205 provided with a lower diaphragm and carrying the internal cone.
• an inlet 208 allows the perfusion of the culture medium and an outlet 209 ensures its evacuation.
• a washer 207 associated with a spring leaf or a return silicone washer allows, by its thickness, the adjustment of the height of the movement printed by the movable part 205.
• a rod or blocking clamp 206 limits the vertical movement by abutting against washer 207.
• the reactor represented in FIG. 6 comprises a vessel 302 "alveolate” which preferably is made of bio-glass, with a truncated-conical alveolus which makes it possible to provide the interface with the internal cone 313 (solid part of the implant , for example the ligaplant) and thus obtain a mechanical stimulation of the culture with a shearing effect which approximates the physiological conditions to which the periodontal ligament is subjected and with a dilation-compression effect allowing a homogeneous diffusion of the culture medium between the rigid walls.
• the size of the device can be adapted to the desired culture surface (frusto-conical), and therefore to the quantity of cells.
• the space between the internal cone 313 and the cell is 0.2 ⁇ 0.1 mm.
• the tank 302 can be inserted into a tank 301, preferably made of polymer and for example of PTFE, which is provided with a cover 303 which is also advantageously made of polymer.
• the tank 301 has a flat bottom so as to allow the possible use of the reactor on a heating table and thus ensuring a homogeneous distribution of the temperature.
• the temperature regulation system can also be directly included in the wall of the tank 301 of the bio-reactor. Again, it may be a water circuit or an electrical resistance or a Peltier effect transistor, etc.
• Two centering pins 308 are inserted in the cover 303 and the body 301.
• inlets-outlets 309 intended to allow the circulation of the medium.
• These inputs-outputs are arranged in vertical lines, 90 ° from each other in plan and alternated to obtain optimal diffusion middle. These input-output are most often two or three.
• the development of vertical slots can also be achieved as well as that of a cell in four quarters.
• Openings 310 are also made in the tank 302 to allow the passage of the pipes and connection fittings or that of the threads possibly allowing locking.
• a flexible and thick lower O-ring 312 allows the amplitude of the vertical movement applied in the middle.
• the latter is preferably applied with an amplitude of 0.1 ⁇ 0.05 mm for a period of 10 seconds.
• the device further comprises, in a nut 307, an implant holder
• a safety key 306 provided on the disinsertion axis 305, makes it possible to block the rotation of the mobile assembly carrying the implant and which comprises the parts 305, 306, 307, 304 and 313.
• a diaphragm 311, associated with the cylinder prevents rotation of the mobile assembly.
• Such a device makes it possible to optimize and equalize the flow of nutrient medium thanks to the multiple inputs-outputs provided.
• the invention can be implemented with different types of implants, of varied shape, structure and composition and for different applications. It is advantageously a dental implant, the root part of which is covered with cells by means of a reactor according to the invention.

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• 2002
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