DE102012101078A1 - Stimulation cell and method for in vitro stimulation of cells or tissues - Google Patents

Abstract

Stimulation cell and method for in vitro stimulation of cells or tissues in a stimulation cell. In the stimulation cell, an interior (10) of the stimulation cell has a carrier (36) which can be colonized by cells or tissue at least over areas of its surface and at least one actuator which is designed to be topically disposed on the surface of the carrier ( 36) a meterable gradient with a growth of the cells or tissue stimulating effect is evoked present. To carry out the method, the steps of introducing a cell substrate or tissue-carrying cell substrate (20) into an interior space (10) of a stimulation cell; inserting a carrier (36) which can be colonized or already populated by the cells or tissue over at least populated areas of its surface, into a receiving device (30) in the interior (10) of the stimulation cell; the introduction of a medium (M) for the physiological care of the cells or tissues and the elicitation of at least one dosable, topical gradient with a cell or tissue stimulating effect on the surface of the carrier (36).

Description

The invention relates to a stimulation cell and a method for in vitro stimulation of cells or tissues.

Bioreactors have been developed, modified and patented since the nineties of the last century as part of the so-called tissue management. These were mainly characterized by the use of fluidics or microfluidics, which attempt to reproduce the otherwise lacking nutrient, active and signaling supply virtually in vitro and thus also implement signal transduction and taxies. The fluidics became particularly important for the cellular colonization of scaffolds. Exemplary here are the DE 39 23 279 and the DE 10 2004 054 125 A1 called. In addition to the delivery of various growth and differentiation factors (gene activation / gene expression), the inherent stimulation principle consists of mechanotransducing effects derived from the fluidic process at the scaffold.

In J Exp Zool. 1990 Feb; 253 (2): 163-76, Harris AK, Pryer NK and Paydarfar D. in "Effects of electric fields on fibroblast contractility and cytoskeleton." a non-fluidic electric field chamber for the alignment of osteoblasts in DC electric fields. The cells align themselves after a certain time at right angles to the field lines and elongate ( ST Curtze (2006, "Tensile Force Microscopy on Osteoblasts", Diss. Human Medicine, University of Marburg: 8 ff. ). This field chamber was used to analyze the mechanotransduction of osteoblasts rather than tissue engineering.

In DE 37 35 702 A1 a method for influencing growth, division and differentiation of cells is presented in a culture container, in which the use of electrical DC fields, changeable in direction and strength, electrical dipole moments of the cells are used in the sense of a spontaneous polarization. In addition to the change in the applied DC field and the electrical surface potential should be changeable. The device for this purpose is based on two parallel, planar plate electrodes, which are electrically insulated from the culture medium or electrically conductive. The area of the plate electrodes corresponds to the base area or at least one inner wall surface of the culture container. The device is used to produce biotechnologically and biomedically important cells, tissues, organs and unicellular organisms as well as biosensors. A spatially directed development of cells or tissues and a colonization of degradable and non-degradable implant surfaces and tissue substitutes is not provided.

In numerous scientific work electrical arrangements, eg. T. in the form of chambers, described with cells and / or microorganisms, which have an analysis of the corresponding influences of electric fields and / or currents to object. Common to them are usually static, invariable arrangements of electrodes, which can only be operated variably by their electrical parameters (magnitude, current direction and frequency). Usually one or two parallel pairs of electrodes, partially orthogonal to each other, are used, which are formed either in or to the medium electrically conductive (current flow) or electrically isolated (voltage / electric field). The pairs are usually in a common plane and act largely planar in their preferred direction. Therefore, these devices are not used for spatial tissue engineering on three-dimensional implant bodies, but primarily for research into the behavior of cells and microorganisms under the influence of defined gradients.

Exemplary are the works of Caubet, R. et al (2004, A radio frequency electric current enhances antibiotic efficacy against bacterial biofilms, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY: 4662-4664) and van der Borden, A. et al. (2007, Prevention of pin tract infection in external stainless steel fixator frames using electric current in a goat model, Biomaterials 28: 2122-2126) cited.

Also known are writings on the stimulatory influence of pulsed electromagnetic or magnetic fields on the growth primarily of bone, cartilage and muscle tissue. Thus was in two review publications of Hungerer, S. et al. (2008, Electromagnetic Procedures in Bone Healing Disorders. Evidence of this Additive Measure, Trauma Occupational Disease 10 [suppl. 2]: 219-225) and Schmidt-Rohlfing, B. et al. (2010, Electromagnetic Fields, Electricity and Bone Healing: What Is Secured? Electromagnetic Fields, Electric Current and Bone Healing - What is the Evidence ?, Z Orthop Accident: 1-6) attempts to summarize the results of experiments on electrical and electromagnetic stimulation in the form of meta-analyzes in order to obtain information about their effectiveness. For the significance and evidence of the procedures to be examined, the evidence of electrical and electromagnetic procedures for bone healing is discussed and assessed on the basis of existing randomized clinical studies.

Unfortunately, the techniques of stimulation (DC / AC electrically conductive, DC / AC electrically isolated, magnetic) were considered equivalent in these studies. A summary of this kind is to be considered with reservations, although a good overview of the clinical trials is given. Apart from the comparability of the above-mentioned methods, the PEMF (Pulsed Electro Magnetic Field) (PEMF) studies are already critical due to the large number of different stimulation parameters and different treatment times. Many of the cited references lack the exact indication of stimulation parameters. It is often incomprehensible which flux densities, frequencies and amplitudes were used.

Table 1 lists commercial stimulation devices with the accompanying technical parameters. Manufacturer Frequency range [Hz] Current [A] Maximum magnetic field density [mT] 1 1-60 0-17 0.001-0.02 2 1-25 - 3-5 3 - - 0.5-5 4 0.5-300 0.001 to 1.2 4.5 5 0.1-40 0-3,5 0.001-10 6 0.5-20 - 2-20 7 0.001 to 38,000 - 15 8th 1-10000 0.00006 to 0.455 To 0.15 9 0.2-20 3-5 0.005 to 0.035 10 2-20 3-5 0-10 11 1-1000 - 100-180 Table 1 Source is a survey on a report by Prof. Gabriel on magnetically induced electrostimulation, Landessozialgericht NRW, Az LSKR 245/00

Table 1 shows that the physical targets differ by several orders of magnitude. The work dealt with the influence of the field quantities on the effectiveness of electromagnetic methods and described above all the low frequency continuous sinusoidal signals in the frequency range below 120 Hz as effective, with a maximum at 15-30 Hz.

Studies with an EBM Level 1 (EBM = Evidence-based Medicine) and numerous other studies with lower EBM levels are available.

Although the literature is numerous and can compete with other additive therapy concepts, such as shockwaves, ultrasound or bone matrix proteins, it is not surprising that electromagnetic therapy has so far not achieved a breakthrough. Due to the large number of commercial devices with very different parameters, the choice of the optimal device is almost impossible for the user. In addition, standardized therapeutic options are lacking because, on the one hand, the fields used by the research teams differ significantly in terms of their physical parameters, and on the other hand, a generally recognized mechanism of action has not yet been described. This also lacks study protocols for the treatment.

In summary, it can be stated that numerous suppressive and stimulative methods and methods have been researched and more or less validated in in vitro and partly also in in vivo studies. Much less could these developments be realized in situ or for in situ applications. There are many experimental studies with very significant results, but hardly any randomized clinical trials.

All of the abovementioned inventions, published in vitro arrangements, stimulation and suppression methods as well as the products in use or sold commercially do not depict the in situ situation of three-dimensional implant shaped bodies in vitro. Also, a gap situation of two opposing interfaces with a more or less large gap is not shown. The in-situ three-dimensional cell / tissue development / adaptation as conduction and / or induction as well as gap-bridging and scaffolding-filling integration through growth or filling in of volume virtually does not take place. In addition, in all these arrangements, the gravitational aspect in cell / tissue development is neglected or abolished by fluid arrangements, although the atypical "flattening" of cultured cells on the bottom of culture dishes or on non-organic membranes is well known.

The spatial distribution of cells / tissues including the extracellular matrix (= ECM) on three-dimensional shaped bodies, d. H. the colonization of implants with preferably autologous cells, and their temporal sequences with respect to the layer formation, crosslinking and ECM, in contrast to the above-mentioned techniques, arrangements and methods requires a transformation of the planar stimulation / Suppressionsmodelle / -anordnungen / -verfahren in the in vitro imaged three-dimensional space in situ. Therefore, the total settlement area has to be resolved topographically into subareas.

In contrast to the fluidic bioreactors, which introduce chemotactic gradients in the form of growth factors, as well as stimulating by the fluidic mechanotransduction of the cells, is an in situ close colonization of three-dimensionally shaped implants or their in vitro simulation for in situ / in vivo stimulation and / or suppression without a real, spatially and temporally resolved, varying application of also applicable in situ gradient, variable in nature, amount, direction and time course, necessary.

At the same time, the process of in situ-proximal colonization of the implant surface with autologous cells not only reduces the risk of inflammation (inflammation) through the formation of biofilms on unoccupied, free surfaces, but also accelerates the secondary anchoring of the implants in situ.

The relevance of using autologous cells to precontain the implants has been demonstrated in experimental monitoring trials to evaluate the bioactivity of implant surfaces. The variance or standard deviation of the adhesion, proliferation, migration and differentiation properties of patient-specific cells with different genetic predisposition or pathologically induced changes (metabolic / circulatory diseases, diabetes, alcohol, nicotine, drug abuse, etc.) is different here bioactive implant surfaces partially more dominant than the variance or standard deviation of the bioactivity of different implant surfaces against an immortalized cell line.

In practice, this means that it is not necessarily just an optimal bioactive surface z. B. for the adhesion of human osteoblasts must, but each individual patient different bioactive surfaces can be optimal. The cost explosion of implantation medicine, possibly accompanied by such an individual medicine due to an exaggerated, individual implant provision on the part of the industry or implant surface modification by the industry in general and / or implantologists, leaves a patient-specific or individual-medically indicated, in vitro evaluated and validated stimulation of autologous , human cells / cell lines appear in vitro or in situ as a cost and risk reducing alternative. This also applies to the use of such arrangements and methods for a patient-specific or individualized stimulation / suppression of differentiation / dedifferentiation and migration / metastasis behavior / potentials of healthy and carcinogenic cells and tissues in oncology.

• – akustische oder mechanische Stosswellen oder Ultraschall (Mechanotransduktion) inkl. Vibration und Fluidik,
• – positive wie negative Gravitation,
• – Spaltsituationen (Kohäsion, Adhäsion),
• – Oberflächenspannung/-energie an der Grenzfläche,
• – elektrische Felder DC/AC (DC = Gleichspannung/AC = Wechselspannung) – isoliert,
• – elektrische Felder DC/AC – leitend,
• – elektromagnetische und/oder magnetische Gleich- und Wechselfelder,
• – Chemotransduktion.

The suppressive and stimulatory methods and methods known from the abovementioned patent and technical literature, some of which are already in practical use, differ in the respective gradients acting on the surfaces, the interfaces or in the moldings:

• - acoustic or mechanical shock waves or ultrasound (mechanotransduction) incl. Vibration and fluidics,
• Positive and negative gravitation,
• - gap situations (cohesion, adhesion),
• Surface tension / energy at the interface,
• - electric fields DC / AC (DC = DC / AC = AC voltage) - isolated,
• - electric fields DC / AC - conductive,
• - electromagnetic and / or magnetic DC and AC fields,
• - Chemotransduction.

The stimulatory and suppressive gradients in bone and soft tissue (bone and soft tissue) management should, in principle, be based on the body's inherent systemic gradients, which can also occur in the organism.

For this reason, it makes little sense to evaluate and validate in vitro signal types, forms and strengths, which are not generated system biologically in situ and, in principle, artificially topically can not be introduced. This decision is based on the fact that the introduction of stimulatory and / or suppressive gradients must always be understood as additives to the body's own healing and growth processes. This additive character is the premise of any method. The additive process must always be within the limits of the body's own system-biological generative process. Furthermore, there are always different cell types in the topical therapy area, which mutually condition and chemotactically influence each other during generative or regressive processes. There is little point in stimulating one cell type only if other cell types are suppressed. The same applies to the neighborhood and the interaction of bone and soft tissue.

The invention has for its object to propose a possibility for topical in vitro stimulation of cells or tissues.

The object is achieved by a stimulation cell for in vitro stimulation of cells or tissues, having a body enclosing an interior, each comprising an upper part and a lower part and at least one actuator for inducing a stimulating effect on at least a portion of the cells or tissue. The stimulation cell is characterized in that in the interior of a carrier is present, which is populated at least over areas of its surface by cells or tissue. The actuator is designed so that it is topically on the surface of the carrier, a dosable gradient with a growth of the cells or tissue stimulating effect is evoked.

The term stimulation is understood to mean any effect on biological processes such as growth, migration or proliferation. Stimulation can also be neutral or negative (suppression). The stimulation cell can therefore also be used as a suppression cell.

Portions of cells or tissues may be spatially on a cell substrate delimable cells or tissue, eg. B. cells or tissues that are carried on a particular sub-area of a cell substrate may be. It may also be certain cell or tissue types that are stimulated.

An actuator is to be understood below as meaning all means by which at least one gradient of a stimulating effect on the cells or tissue can be caused. Stimulatory effects may be beneficial (positive), suppressive (negative) or neutral. Stimulating effects may preferably be caused by electrical, electromagnetic, magnetic, chemical or physical stimuli. The actuator is preferably designed in such a way that the gradient of the stimulating effect (stimuli) caused by it is spatially defined, directed and has an intensity-variable effect on the growth of the cells and tissue. The gradient may be in its properties, such. As its waveform, strength, magnitude, intensity, direction and extent, and caused variable (metered).

The carrier is preferably a shaped body, for. B. an implant molding (Scaffold). The carrier can basically consist of any material. However, carriers are preferably shaped bodies of at least one biodegradable material, of at least one non-biodegradable material or of a combination of these materials or tissue substitutes, the latter being given a shape.

Under the surface of the support is both an outer, an outline of the molding body defining surface to understand, as well as an inner surface, as it is found in particular in moldings of porous material or moldings, having depressions and molds.

The terms bio-degradable and degradable mean that the material in question can be degraded by biochemical reactions of the tissue (eg enzymatically) and optionally in interaction with biophysical processes.

The upper part and the lower part of the body can in other embodiments of the stimulation cell according to the invention also similar, z. B. two half-shells, or be firmly connected to each other and a closed body, z. As a cylinder form. By further design forms, the lower and upper part can be designed differently, so that a classic separation in lower and upper part is resolved.

In an advantageous embodiment of the stimulation cell according to the invention at least one media supply and at least one media removal on at least one side of the interior for the supply and discharge of a medium is present.

By the media supply and the removal of media, it is advantageously possible to add chemical stimulants in a defined manner to the medium and thus to stimulate the cells or tissue. Such stimulants can be chemical substances that have a stimulating effect on growth, such as growth factors, nutrients, hormones and energy equivalents. Also, a monitoring of the processes in the stimulation cell by z. B. allows an analysis of the supplied and the discharged medium. The medium is preferably electrically conductive and serves for the physiological supply of the cells or tissue.

Hereinafter, by the term of the growth of cells or tissues, all growth-related processes, such as. Cell migration, differentiation and proliferation. In addition, processes such as the death and degradation of cells (apoptosis, suppression) can be stimulated with the stimulation cell according to the invention and, optionally, can also be analyzed.

The cells or tissues may be present on as well as in a cell substrate other than the carrier and are therefore carried by the cell substrate. The cell substrate is arranged in the interior of the stimulation cell.

A cell substrate may be any material or mixture of substances through which cells are provided, for example colonized or inoculated nutrient media such as agarose, alginate, mono- and co-cultures.

The stimulation cell of the invention is also useful for stimulating biofilms formed by, for example, bacteria, fungi, algae and protozoa. In addition, means may be provided by means of which the effects of the stimulations can be detected and analyzed.

In the course of growth, the carrier and cell substrate can be covered with a large number of cells, between which ECM can also be formed or tissue can differentiate. In the following, simplification of cells is mentioned, but all possible cell biological structures and units as well as single- and multicellular organisms should be included.

In a preferred embodiment of the stimulation cell according to the invention, at least two electrodes are present. These are designed to generate spatially and temporally defined electrical and / or electromagnetic fields with variable shape, magnitude, direction and intensity. The electrodes are selected from a group comprising electrically conductive electrodes against the medium, electrically insulated electrodes against the medium and hybrid electrodes formed from electrodes and electrically conductive electrodes electrically insulated from the medium. Hybrid electrodes represent a combination of electrically isolated and electrically conductive electrodes, either in the sense that portions of the hybrid electrode are isolated against the medium and other portions are not isolated or that both types of electrodes are present separately, but only in contrast to the separate variant of the two types of electrodes an identical control possibility exists for both types of electrodes. The electrodes can be segmented.

In further embodiments of the stimulation cell, cluster electrodes may also be present which are formed from electrodes which are arranged in grid form and / or electrodes which can be driven by signal technology.

It is also possible that at least one magnetic field generator is assigned to the stimulation cell as an actuator, so that the stimulation cell is arranged at least over regions in a magnetic field generated by the magnetic field generator. The magnetic field generator may be movable along or around the stimulation cell. The magnetic field generator may be, for example, a magnetic coil.

For example, the magnetic field generator as an actuator, but also other actuators, with respect to the stimulation cell and against the carrier to be movable, whereby by means of an actuator gradient from changing directions can be generated or a gradient over the surface of the carrier can be guided.

It is also possible that a hybrid actuator is present, through which different gradients can be produced simultaneously or consecutively from one another. For example, can be caused by a piezo stack both electrical and mechanical gradients. The gradients may be of the same physical or chemical nature, e.g. B. two electromagnetic gradient or two growth factors. The gradients can also be of different nature, e.g. For example, an electrical gradient and a mechanotransducing gradient may be caused by a hybrid actuator.

The actuator is advantageously such that the stimulating effect produced by the actuator is assigned to certain subregions of the cell substrate and limited to these. Depending on the location of these subregions and the expression of the stimulating effect, a directed colonization of the carrier is stimulated only on a partial surface of a surface of the carrier.

For this purpose, the electrodes may be formed segmented. By segmentation is a generation of electromagnetic or magnetic fields with spatially determined expression, z. B. the course of the field lines and the distribution of field strengths, spatially significantly resolved as possible with unsegmented electrodes.

In the interior, at least one receiving unit for receiving the carrier or a cell substrate may be arranged. Preferably, the receiving unit is configured such that between a surface of a carrier located in the receiving unit and a surface of a cell substrate located in the receiving unit, a gap is adjustable by which the surfaces of the carrier and the cell substrate are separated from each other. In a further embodiment, at least two receiving units may also be arranged such that a gap is adjustable between a surface of a carrier located in a receiving unit and a surface of a cell substrate located in another receiving unit, by which the surfaces of the carrier and the cell substrate are separated from each other , The carrier and the cell substrate may also be held together in a correspondingly formed receiving device. Preferably, at least one of the receiving devices allows positioning of the carrier and / or the cell substrate in the interior space.

A preferred embodiment of the stimulation cell according to the invention is given by the fact that the carrier and at least one gradient are movable relative to each other. This can be made possible by relative movements of the carrier, the actuator or as a combined movement of the two.

In a further embodiment of the stimulation cell according to the invention, the carrier is temporarily arranged with at least one magnetic body or a magnetizable body in direct or indirect contact. This magnetic body or magnetizable body lies at least partially in the field lines of a primary magnetic field, so that in the magnetic body or the magnetizable body by the primary magnetic field, a secondary magnetic field is variable. Under a change of the secondary magnetic field and its generation, for. B. at a magnetization of a magnetizable body understood. The secondary magnetic field can be used, for example, as a source of induction.

A gap in the sense of this description is a spatial gap. This is when the carrier object and the cell substrate are actually arranged at a distance from one another. It is preferred that at least one of the receiving devices enables positioning of the carrier and / or the cell substrate in the interior space. The gap has a gap width, which is preferably adjustable up to a gap width of 3 mm. Through the gap is an in situ situation between a surface of a carrier, for. B. an implant molding, and a cell substrate, such as a tissue replicated.

In order to enable an analysis of the processes in the stimulation cell, the electrodes can be designed as measuring means for measuring electrical quantities. It is also possible that further measuring means for measuring physical quantities are arranged.

It is very advantageous if an evaluation, storage and control unit is present, which is connected to the electrodes and other measuring means. The evaluation, storage and control unit can be connected to a database.

Physical stimuli such as pressure or accelerations can be coupled in, if, in an advantageous embodiment, the stimulation cell is connected to a controlled drive by means of which the stimulation cell can be moved in a controlled manner.

The object is further achieved by a method for in vitro stimulation of cells or tissues. The essential steps of the method according to the invention are the introduction of a cell substrate or tissue-carrying cell substrate into an interior of a stimulation cell, followed by the insertion of a carrier which is populated or already populated by at least settleable areas of its surface by the cells or tissue, in a receiving device in the interior of the stimulation cell. Subsequently, a medium for physiological supply of cells or tissue is introduced into the stimulation cell. Furthermore, at least one meterable topical gradient is produced with a cell or tissue stimulating effect on the surface of the carrier.

The stimulating effect can be assigned to certain subregions of the cell substrate and caused to be limited to these. It is advantageous if a spatially directed colonization of certain parts of the populated areas, z. B. across a gap, is stimulated.

The medium can be fed under control to renew the medium regularly. Various media can be supplied.

The stimulating effect is selected from the generation of electromagnetic fields, electric fields, magnetic fields, the addition of chemical compounds and the coupling of physical stimuli.

In an advantageous embodiment, measured variables whose measured values are variable as a result of reactions of the cells and tissue to the stimulating effect are recorded and stored. It is possible to record and store initial measured values of at least one measured variable at an initial measuring time and to use the initial measured values as reference measured values for measured values recorded at later measuring times.

It is also possible for information about the growth of the cells or tissue to be derived from the changes in the measured values acquired and stored at different measurement times. The measured values can be recorded time-resolved or spatially resolved or temporally-spatially resolved. In addition, the acquired measured values can be graphically displayed and the information about the growth of the cells or tissue can be derived from the graphical representation. A comparison of the information with a database is possible.

Advantageously, control signals for controlling the actuator can be generated on the basis of the measured values in order to change the gradient. This makes it possible to respond specifically to the growth of cells or tissues.

In a further embodiment of the method according to the invention, a gap with a gap width can be set between the cell substrate and the carrier.

By means of the invention, a test arrangement with opposing boundary layers including gap region is provided in which a multidimensional method for stimulating in vitro cell / tissue growth in a definable interface situation between a tissue-like region and a region in the form of degradable or non-degradable implant surfaces and / or Tissue replacement materials can be realized. In principle, it is intended to simulatively evaluate and validate medically applicable methods for their bioactive and / or antiinflammatory effects on human cells (osteoblasts, endothelial cells, squamous epithelial cells, fibroblasts, etc.) as well as bacteria, fungi and other microbial organisms capable of forming a biofilm, and stimulatively and / or suppressively applied to the biological in vitro analogous to the in situ situation and / or in vivo in the current situation in situ.

• in allen räumlichen Dimensionen drehbar sein, um gravitative Aspekte des Zell- und Gewebewachstums auszuschließen,
• definiert wipp-, kipp-, taumel-, ping-pong- und kreisschüttelbar sein, um Zell- und Gewebewachstumsprozesse sowie auch die Biofilmentwicklung unter normalen, alltäglichen und/oder definierten Körperbewegungssituationen, wie beispielsweise bei der Physiotherapie simulieren zu können,
• definierten Gradienten (beispielsweise Druck, Kraft und Oberflächenspannung) aussetzbar sein, die körperlichen Belastungssituationen in situ vergleichbar sind,
• über Aufnahme- und Entnahmesysteme in Gestalt von Fassungen, Fixierungen und Aufnahmeräumen für jeweils unterschiedliche, austauschbare Träger, z. B. Implantatformkörper und Gewebeersatzstoffe, sowie für unterschiedliche Mono- und Kokulturen in Gestalt von beispielsweise Alginat- oder Agaroseblöcken besitzen,
• über mindestens eine Medienzuführung und eine Medienabführung für die generelle sowie chemotaktisch stimulative Versorgung der Zell- und Gewebewachstumsprozesse sowie auch die Biofilmentwicklung mit Nähr- und Sauerstoff für in vitro Langzeittest sowie für ein mikrobiologisches und/oder biochemisches Mikrotröpfchenmonitoring verfügen,
• einen Höhen-, Breiten- bzw. Abstandsverstellmechanismus für unterschiedlich dicke bzw. große, austauschbare Implantate und/oder Gewebeersatzstoffe (Träger) sowie für die Einstellung des definierten Abstands zu einem der Trägeroberfläche gegenüberliegenden Zellsubstrat sowie zu den Aktoren besitzen,
• über definierte fixe und/oder räumlich veränderliche, austauschbare Elektroden- und/oder Spulenanordnungen inklusive zu applizierender Stimulationsprofile (workflows) verfügen
• sowie, gegebenenfalls optional, mittels der erfindungsgemäßen Stimulationszelle ein biophysikalisches, nichttestabbrechendes Monitoring-/Controllingmessverfahren durchführbar sein, welches komplementär und komparativ zu den mikrobiologischen Standardtest- und Prüfverfahren benutzt werden kann, um Bibliotheken zu erstellen und den Verlauf zu dokumentieren, mit dem Ziel, auf zeit- und kostenaufwändige mikrobiologische Testverfahren verzichten zu können.

In order to come as close as possible to the in situ situation to be simulated, the stimulation cell should:

• be rotatable in all spatial dimensions to exclude gravitational aspects of cell and tissue growth,
• defined rocking, tilting, tumbling, ping-pong and kreisschüttelbar be able to simulate cell and tissue growth processes as well as the biofilm development under normal, everyday and / or defined body movement situations, such as in physiotherapy,
• defined gradients (eg pressure, force and surface tension), which are comparable to physical stress situations in situ,
• about receiving and removal systems in the form of sockets, fixations and receiving spaces for each different, interchangeable carrier, eg. B. implant shaped bodies and tissue substitutes, as well as for different mono- and co-cultures in the form of, for example alginate or Agaroseblöcken own
• have at least one media feed and a media removal for the general and chemotactically stimulative supply of the cell and tissue growth processes as well as the biofilm development with nutrient and oxygen for in vitro long-term test and for a microbiological and / or biochemical microdroplet monitoring
• have a height, width or distance adjustment mechanism for different thicknesses or large, replaceable implants and / or tissue substitutes (carrier) and for setting the defined distance to a cell substrate opposite the carrier surface and to the actuators,
• have defined fixed and / or spatially variable, exchangeable electrode and / or coil arrangements including stimulation profiles (workflows) to be applied
• and optionally optionally, by means of the stimulation cell according to the invention, a biophysical, non-test-abortive monitoring / controlling measurement method can be carried out, which can be used complementary and comparative to the standard microbiological test and test methods to create libraries and document the course, with the aim of to be able to do without time-consuming and costly microbiological test procedures.

Background of the invention is an in vitro / in vivo / in situ simulation, evaluation and validation of stimulation and suppression methods for the in vitro or accelerated in situ colonization of implants with autologous, human cells and tissues, eg. B. for improved and / or accelerated ossification, osteogenesis and / or osseointegration of carriers of different developers and manufacturers with the lowest possible in vivo use.

The aim of the invention is to provide multidimensional, static or dynamic stimulation with optional monitoring of the cell / tissue by means of an actuator and optional sensor process arrangement for generating and optionally measuring biophysical / (bio) electrical / (bio) magnetic parameters. / microbial growth in in vitro tissue management procedures on or in functional, bioactive carrier surfaces and / or tissue substitutes.

For this purpose, topical gradients are applied in vitro to human, autologous, immortalized and / or altered cells and tissues, which can be varied in type, signal shape, direction and magnitude and in spatial-temporal progression, preferably on three-dimensional carriers for stimulation of the cell and tissue Tissue growth and biofilm formation processes by microbial organisms. The cells or tissue are already present on the carrier or reach an area of action of a gradient caused on the surface of the carrier.

• – eine in vitro Simulation von physiologischen oder additiv applizierten in vivo Stimulationsprozessen des Zell- und Gewebewachstums sowie der Biofilmbildung durch mikrobielle Organismen,
• – eine Evaluierung und Validierung von topischen Gradienten an Oberflächen und/oder in und an Scaffolds in vitro,
• – eine Optimierung des Verfahrens und Anpassung der Aktoranordnung in vitro an die in vivo bzw. in situ Situation,
• – eine Extrapolation der in vitro Simulation auf die in situ Situation mit, gegebenenfalls auch nachgelagerten, ex vivo und insbesondere nachgelagerten in vivo Verfahrenskomponenten,
• – eine definierte Besiedlung von Implantatkörpern beziehungsweise deren Oberflächen beziehungsweise von resorbierbaren und/oder nicht resorbierbaren Scaffolds mit autologen Zellen und Geweben zur individualmedizinischen Optimierung der Primär- und Sekundärverankerung von Implantaten und zur Vermeidung von Inflammationen durch mikrobielle Biofilmbildung auf unbesiedelten Trägerbereichen wie Implantatoberflächen oder Scaffoldinnenräumen,
• – eine gezielte Beeinflussung von Adhäsion, Proliferation, Migration oder Differenzierung/Dedifferenzierung von Zellen, Zellverbänden, Zellschichten sowie der Entwicklung der extrazellulären Matrix und
• – eine gezielte Beeinflussung der Gewebsstruktur/-geometrie im Sinne einer definierten Zellausrichtung mit anschließender Ausbildung eines gerichteten Gewebeverbunds.

It takes place

• An in vitro simulation of physiologically or additively applied in vivo stimulation processes of cell and tissue growth as well as biofilm formation by microbial organisms,
• - evaluation and validation of topical gradients on surfaces and / or in and on scaffolds in vitro,
• An optimization of the method and adaptation of the actuator arrangement in vitro to the in vivo or in situ situation,
• An extrapolation of the in-vitro simulation to the in situ situation with, if appropriate also downstream, ex vivo and in particular downstream in vivo process components,
• A defined colonization of implant bodies or their surfaces or resorbable and / or non-resorbable scaffolds with autologous cells and tissues for individual medical optimization of the primary and secondary anchoring of implants and to avoid inflammation by microbial biofilm formation on uninhabited support areas such as implant surfaces or scaffold interiors,
• Targeted influencing of adhesion, proliferation, migration or differentiation / dedifferentiation of cells, cell aggregates, cell layers as well as the development of the extracellular matrix and
• - A targeted influencing of the tissue structure / geometry in the sense of a defined cell alignment with subsequent formation of a directed tissue composite.

It is possible to extrapolate an arrangement of the actuator or several actuators found by an in-vitro simulation as well as a workflow to situations and applications in vivo. In particular, such arrangements and workflows may be transferred and applied to wearable or implantable devices.

According to the arrangement, the outer shape of the stimulation cell is to be designed in its dimensions and its shape as well as its actuator arrangement such that the carrier can be accommodated in its in-situ dimension. As a rule, it is designed as a cuboid with receiving devices for the carrier. In the case of rotationally symmetrical or spherical carriers, a cylindrical, hemispherical or spherical shape and combinations thereof are advantageous.

According to the arrangement, the outer stimulation cell is designed to be fixed relative to the inner support. The enclosed by the walls of the stimulation cell carrier can be mounted fixed or movable. For special hollow body, the arrangement may also be formed inversely, d. H. it can parts of the stimulation cell in z. B. cavities of the carrier protrude.

According to the arrangement, the at least one actuator is arranged such that it can reach the overall surface of the carrier, preferably dividing into sub-areas defined by biostimulatory or suppressive gradients in the direction, magnitude and signal shape, without affecting individual subareas with divergent, less biostimulatory or internal regions suppressive or even the application benefits according to the invention counteracting gradient is acted upon. For planar surfaces, the subregions can be maximally expanded or coincide.

According to the arrangement, topical electrical, electromagnetic and mechanotransduktive gradient / stimuli are applied in vitro via actuators within and outside the stimulation cell, which are arranged at least in one dimension on the walls or in the center of the stimulation cell. Electrical stimuli are introduced via spatially and temporally stimulation regions associated electrode arrangements. Chemotactic gradients are introduced fluidically, preferably via the media supply. Gravitational, mechanotransduktive and acceleration gradients can be introduced into the stimulation cell via a corresponding, preferably rotary, tiltable and / or swingable, outer support of the stimulation cell. Surface tension and surface energy gradients can be achieved by means of magnetizable elements which can be placed temporarily on or in three-dimensional implant shaped bodies, for example As layers, sheaths or cylinders, via defined associated magnetic actuators or mechanical stress devices are applied.

In general, the electrical or electromagnetic, effective actuators in the form of compared to the conductive medium within the stimulation cell electrically conductive and / or electrically insulated electrodes and magnetic field coils, for. B. in pairs on the inner or outer walls of the stimulation cell facing each other, arranged. The coil arrangement can also be designed to surround the interior in at least one plane. The field geometry of the magnetic fields caused by the magnetic field coils can be varied.

Electrodes can be designed as electrically conductive, non-insulated electrodes, whereby a flow of current between at least two electrodes and through the medium is made possible. By means of non-isolated electrodes measurements based on current flow can be carried out. If the electrodes are designed to be electrically insulated, these can be used to generate electrical and / or electromagnetic fields in the interior and to use them for the measurement. The electrodes may be formed in various shapes, e.g. B. stripe, plate or punctiform or combined to form so-called clusters. The electrodes may be fixed or freely positionable in the interior. This advantageously provides high flexibility in the design of the measuring arrangements. There may be a plurality of non-insulated electrodes, a plurality of insulated electrodes and hybrid electrodes. It is advantageous if the shape, arrangement and the manner of generating the electromagnetic fields allows a spatially resolved detection of the measured variables. The electrodes can also be segmented. This can be achieved, for example, by a spatially defined arrangement of a number of electrodes, wherein a location of the detection of the measured variables can be derived from the differences in the measured variables per electrode and the knowledge of the arrangement of the respective electrodes.

According to the arrangement, the biostimulative and / or suppressive gradient in the form of individual gradients, gradient overlays and / or gradient deflection by auxiliary gradients / fields in amount, direction and shape can be topically defined and applied on any subarea of the cell substrate.

According to the arrangement, the distribution and placement of the actuators, for example axially and / or planar symmetrically, in at least one and preferably in all three spatial axes according to the method enables a topically defined application of the (bio) stimulatory gradients to defined partial areas of the support. Thus, hitherto known or new stimulation methods, which are preferably effective in one dimension, can be applied to three-dimensional carriers by means of the overall arrangement as well as a spatially-temporally topically transforming signal and actuator control.

According to the arrangement, the fixation of the three-dimensional support within the stimulation cell can also be ensured via metrological aids, the topography or its partial surface resolution of the shaped body and its spatial allocation to the distribution and design of the actuators, by placing the sub-coordinate system of the support in the main coordinate system of the stimulation cell in the downstream Evaluation, storage and control unit integrated and calculated as dual surface system (inner surface of the cell to the outer surface of the carrier).

According to the arrangement, it can be useful for the receiving device of the carrier to be configured such that the carrier itself acts as part of the stimulation actor or the optional monitoring sensor system. Electrical stimuli may be introduced as an electrode using, for example, a conductive carrier or a conductive portion of the carrier. Likewise, the conductive support or its portion may also serve as a measuring electrode for monitoring. Mechanical stimuli can also be introduced, for example, by direct local influence (for example, by movements, rotations or vibrations) of the wearer.

According to the arrangement, it can be useful for the carrier to be movably arranged at its fixation points with the stimulation cell, so that a rotation or displacement of its surface is possible with respect to the stimulating fields, with the aim of covering a larger part or even the entire surface of the carrier to bring the direction of the stimulating fields or to align to different gradients of these fields.

According to the arrangement, it may be useful for the actuator to be able to apply several types of stimuli at the same time. Thus, piezoelectric stacks designed as electrodes could at the same time apply electrical and mechanical stimuli or magnetically moved bodies or magnetizable layers simultaneously with magnetic and mechanical stimuli.

To excite the arrangement, both defined individual frequencies and whole bands of frequencies in the sense of a frequency sweep and direct signals can be used. The exciting signals can also be varied in their amplitudes and their signal shape. The electrodes provided in the arrangement can be varied and distinguished in several respects. On the one hand with regard to their connection with a conductive nutrient solution / a medium in conductive (wet) and non-conductive (dry) electrodes in the form of electrically insulated electrodes as well as electrically conductive, galvanic electrodes, on the other hand with respect to their geometry, eg. B. in point, line, and surface electrodes, and their arrangement with each other, z. B. in single electrode arrangements, raster arrangements and free arrangements. Furthermore, theoretically, any combination of the intended electrodes can be used for excitation. For this purpose, the actuators are advantageously implemented with one another or with different individual electrodes of a grid above the surface or under a seeded carrier (gradient generation along the carrier) or a selection of electrodes of the grid.

The invention will be explained in more detail with reference to embodiments and figures. Show it:

1 a first embodiment of a stimulation cell according to the invention in a side sectional view;

2 the first embodiment of the stimulation cell according to the invention in the plan view of the lower part of the stimulation cell;

3 the first embodiment of the stimulation cell according to the invention in the plan view of the stimulation cell with attached upper part;

4 a first embodiment of an actuator with electrically insulated and electrically conductive electrodes;

5 a second embodiment of an actuator with electrically insulated and electrically conductive electrodes as a hybrid electrode;

6 a third embodiment of an actuator with electrically insulated and electrically conductive electrodes as a cluster electrode;

7 a second embodiment of the stimulation cell according to the invention with a vertically arranged magnetic field coil;

8th A third embodiment of the stimulation cell according to the invention with a horizontally arranged magnetic field coil;

9 A fourth embodiment of a stimulation cell according to the invention with a horizontally arranged magnetic field coil;

10 a fifth embodiment of a stimulation cell according to the invention in a lateral sectional drawing with receiving device;

11 a sixth embodiment of a stimulation cell according to the invention in a side sectional drawing with receiving device;

12 a seventh embodiment of a stimulation cell according to the invention with a planar magnetic body in a sectional side view;

13 an eighth embodiment of a stimulation cell according to the invention with a planar magnetizable body and gap in a side sectional view;

14 a ninth embodiment of a stimulation cell according to the invention with an arcuate magnetizable body in a side sectional view;

15 A tenth embodiment of a stimulation cell according to the invention with an arcuate magnetizable body and gap in a side sectional view;

16 an eleventh embodiment of a stimulation cell according to the invention in an exploded view and

17 an array of six stimulation cells according to the invention in an exploded view.

A first embodiment of a stimulation cell according to the invention 1 has as essential elements one from a lower part 11 and a top 14 existing and an interior 10 enclosing body, as well as actuators acting electrodes 1 acting as electrically isolated electrodes 1a on outsides of the walls of the base 11 and the shell 14 , as well as electrically conductive electrodes 1b on the insides of the walls of the base 11 and the shell 14 are arranged. One as a carrier 36 serving implant molding (not shown) of a biodegradable material is in the interior 10 arranged. In a side wall of the base 11 is a media feed 12 and in another wall a media drain 13 for the supply and removal of an electrically conductive medium M (only indicated) in and out of the interior 10 available. Thus, the stimulation cell can be connected to a pump and reservoir as well as to a microdroplet removal system (all not shown) to ensure the supply of nutrients, oxygen and / or signal to the cells and tissues and to target other biological organisms into the cleft area can. In addition, with such an arrangement, a defined pressure and flow load situation in the gap region can be generated temporarily or periodically or permanently, for example for mechanotransduktive simulation of stress and / or movement situations. Via the medium M, it is possible to use defined biologically and / or biochemical and / or chemical substances, substances, elements, isotopes and / or chemical compounds.

Out 2 it can be seen that all the electrodes 1 are executed rectangular and planar and respectively over areas of the inner and outer walls of the lower part 11 and the shell 14 (please refer 3 ) pass. In abutting areas of the lateral walls of the lower part 11 are blind holes, each with an internal thread 15.1 present, which is a detachable attachment of a shell 14 (please refer 3 ) by means of screw connections. The carrier 36 is not shown for reasons of clarity.

The stimulation cell with attached upper part 14 is in 3 shown. Corresponding to the blind holes with internal thread 15.1 (please refer 2 ) are in the shell 14 Through holes 15.2 available, by the screws (not shown) pluggable and in the blind holes with internal thread 15.1 are rotatable. Shown are the electrically insulated electrodes 1a of the top 14 and the side walls of the base 11 , as well as media feeder 12 and media removal 13 ,

In 4 Two actuators are shown, each with an electrically isolated electrode 1a and an electrically conductive electrode 1b exhibit. The electrically conductive electrode 1b is from the electrically isolated electrode 1a surrounded and electrically isolated from each other. Both electrodes 1 are unsegmented and can be called global electrodes. The square-shaped actuator can, for example, in stimulation cells according to 1 to 3 at the base of the lower part 11 and / or the top 14 be arranged. The rectangular shaped actuator can be attached to the side walls of the base 11 and / or the top 14 be arranged.

A hybrid electrode comprises according to the in 5 shown embodiment, an electrically isolated against the medium M electrode 1a and an electrically conductive electrode 1b , each with connections 18 are controllable. Such a hybrid electrode may be in the interior 10 a stimulation cell, whereby arranged on the outer walls of the stimulation cell electrodes 1 (see eg 1 ) are replaceable or suppliable.

An electrode designed as a cluster electrode is in 6 shown schematically. The cluster electrode consists of a number of individual electrically conductive electrodes 1b , which are spaced apart, arranged in an array. The array (cluster electrode assembly) is from an outer electrode 16 (global electrode). Between the outer electrode 16 and the array is a border 17 designated area. The border 17 may be formed either as an electrically insulating region or as a ground reference electrode. Each of the electrodes 1 of the array and the outer electrode 16 has one connection each 18 on and is over a connection line 60 with an evaluation, storage and control unit 65 connected (simplified).

In further embodiments of the stimulation cell according to the invention, the array can also be of electrically insulated electrodes 1a or be formed by hybrid electrodes.

A counter electrode to a cluster electrode may be another cluster electrode, a flat electrically insulated electrode 1a , a flat electrically conductive electrode 1b (Global electrodes) and a combination of the aforementioned electrodes 1 be. Several electrodes of a cluster electrode can be driven together.

The electrodes 1 may also be arranged in the form of orthogonally isolated stripe stripe electrodes and as a dot-ring electrode cluster (not shown).

Through the evaluation, storage and control unit 65 are the electrodes 1 individually or in groups controllable and frequency-based to stimulate the migration, adhesion, proliferation, differentiation and development of ECM, formation of monolayers and multilayers of cells, maturation or biofilm formation, suppression and other cellular processes.

In 7 a stimulation cell is shown, in a running in the vertical direction of the stimulation cell level on four sides each have a magnetic field coil as a respective magnetic field generator 19 assigned. The stimulation cell and the magnetic field generator 19 are spaced apart so that the stimulation cell at least partially in each of the by the magnetic field generator 19 generated magnetic fields.

Accordingly, the in 8th designed stimulation cell, the magnetic field generator 19 are assigned, which are arranged in a plane extending in the horizontal direction of the stimulation cell. The magnetic field generator 19 are through the evaluation, storage and control unit 65 (not shown) controllable. The magnetic fields are controlled in terms of their parameters such as geometry and / or field strengths varied.

An arrangement of a magnetic field generator completely enclosing the stimulation cell in a plane 19 is in 9 shown. Combinations of the aforementioned magnetic field generator 19 are possible in further versions of the stimulation cell.

The stimulation cell may be in communication with a controlled drive (not shown) and may be movable by the controlled drive. This can cause a relative movement of the wearer 36 be effected to the actuator and a gradient generated by the actuator.

A further embodiment of the stimulation cell according to the invention is in 10 shown schematically. In the interior 10 is a cradle 30 for receiving and fixing a carrier 36 in the interior 10 available. The cradle 30 is symmetrical and allows the recording and fixation of two carriers 36 each in the form of a planar shaped body. The cradle 30 is adjustable via each adjustment range along all spatial axes, so that the carrier 36 be positioned within a given by the adjustment space space. The interior 10 is by a trough-shaped, open to one side lower part 11 , as well as the open side of the base 11 covering top 14 enclosed. For reasons of representability, the stimulation cell is shown edgewise. Between the shell 14 and the lower part 11 is a circumferential gap for gas exchange 21 available. On a wall of the lower part 11 is on the outside of the wall an electrically insulated electrode 1a and on the inside an electrically conductive electrode 1b available. The cradle 30 protrudes orthogonally from the wall of the base 11 in the direction of the top 14 and spans the interior 10 , Along this direction, the individual elements of the stimulation cell will be described below. At a distance and parallel to the wall is a carrier 36 held. On its side facing away from the wall is parallel to the carrier 36 a cell substrate 20 present, that of the carrier 36 through a constant gap 55 is disconnected. The carrier 36 opposite side of the cell substrate 20 is a flat electrically conductive center electrode 32b facing and spaced from this. The electrically conductive center electrode 32b is an electrically isolated center electrode 32a facing and from this by an isolation 33 separated. The electrically isolated center electrode 32a follow an isolation 33 , an electrically conductive center electrode 32b , a cell substrate 20 , A gap 55 , A carrier 36 , an electrically conductive electrode 1b and an electrically insulated electrode 1a , wherein the electrically conductive electrode 1b and the electrically insulated electrode 1a through an isolation 33 are separated. The described arrangement of elements follows the upper part 14 , The electrically isolated center electrode 32a and the electrically conductive center electrode 32b are about connections 18 controllable. In the walls of the lower part shown above and below 11 are each an electrically conductive electrode 1b and an electrically insulated electrode 1a arranged. In the upper wall is also a media feeder 12 and in the bottom wall a media discharge 13 available. The cradle 30 is also adjustable in itself, so that a gap width of the gap 55 is adjustable. The gap width is adjustable in the embodiment up to 350 microns, it can be adjusted in other versions to 3 mm.

A sixth embodiment of the stimulation cell according to the invention comprises according to 11 an upwardly open lower part 11 on, on its lower wall and on the side walls in each case an electrically insulated electrode 1a and an electrically conductive electrode 1b is arranged. Each side wall has a media feeder 12 and a media transfer 13 on. In further embodiments of the stimulation cell, other numbers and / or arrangements of the media supply can also be used 12 and the media removal 13 to be available. The interior 10 is through the shell 14 completed and over a horizontally extending gap for gas exchange 21 connected to the environment. The top 14 is without electrodes 1 executed. In the field of insulation 33 is a border formed as a ground reference electrode 17 available. The embodiment of the recording device 30 and the other elements of the stimulation cell corresponds 10 ,

The carrier 36 may also be designed non-planar in other embodiments. The gap width of the gap 55 may also be variable over its extent.

The geometry and position of the cell substrate 20 can the geometry and position of the carrier 36 to ensure a defined gap 55 be adjusted.

The geometry of the stimulation cell may be in the design of its upper part 14 and bottoms 11 the geometry and position of the beams 36 be adjusted. The upper and lower parts 14 . 11 may have a cuboid, cylindrical, spherical and / or hemispherical shape and all the composite shapes and their combinations.

The carriers 36 can in the interior 10 concerning the upper and lower parts 14 . 11 be arranged to be movable. A the carrier 36 geometrically adaptable arrangement of the cell substrate 20 may also be advantageous. In the stimulation cell can also be several carriers 36 be arranged.

In 12 is a stimulation cell, as in 1 is described with a recording device 30 shown in the spaced apart two carriers 36 in each case a fixing device 31 are held. The interior 10 is filled with a medium M in which cells are suspended. Between the carriers 36 is a magnetic body 34.1 arranged in the form of a magnetic plate. The magnetic body 34.1 is through an isolation 33 enclosed, the contact of the medium M with the magnetic body 34.1 prevents possible cytotoxic effects of the material of the magnetic body 34.1 to prevent. The carrier arranged above arranged 36 serves as an abutment for the magnetic body 34.1 and the isolation 33 and is not populated (passive carrier 36 ). The arrangement shown, in addition to a stimulation by means of a magnetic field and a force on one side of the carrier 36 applied.

In further embodiments of the stimulation cell, both carriers 36 be colonized or already seeded (active carriers 36 ) in the interior 10 be introduced.

13 shows the stimulation cell as in 1 , with a cradle 30 with fixators 31 in which between two mutually parallel straps 36 a magnetizable body 34.2 is arranged. Between the carriers 36 is an isolation 33 for electrical insulation and to prevent cytotoxic effects of the magnetizable body 34.2 available. Each of the carriers 36 is on the magnetizable body 34.2 one side away, each with a gap 55 spaced, cell substrate 20 assigned.

Depending on a variation of 12 show the stimulation cell shown 14 and 15 , In 14 is a semicircular arched beam 36 the cradle 30 held. The carrier 36 is over its distal areas in the fixator 31 held while under a central area of the carrier 36 a dome-shaped, upwardly arched structure of several layers is present. Under the carrier 36 are also a correspondingly curved magnetizable body 34.2 , an electrically conductive center electrode 32b and an isolation 33 arranged.

In an embodiment according to the 15 are above the arched, central area of the vehicle 36 in addition a likewise arched gap 55 and a cell substrate 20 available.

As in the 14 and 15 shown, a magnetizable body 34.2 be arranged. Will this arrangement with magnetic field generators 19 combined, a magnetic gradient can be generated by means of magnetic fields with a defined variable field geometry, which, mediated via the magnetizable body 34.2 , on the carrier 36 can work. So that can be on the surface of the carrier 36 Voltage states are generated all the way to vibrations when the magnetizable body 34.2 is at least in a partially non-homogeneous field area.

In other embodiments may also be a magnetic body 34.1 or a combination of at least one magnetic body each 34.1 and a magnetizable body 34.2 be arranged.

In all embodiments of the stimulation cell, piezo stacks formed as electrodes can simultaneously generate electrical and mechanical stimuli.

As in the 16 and 17 illustrated, the stimulation cell may also be formed non-fluidically as a static system, but in which also a defined gap situation with gravitationally positive as well as negative gradient can be generated.

According to 16 is between a in a lower receiving unit for carriers with fixing / Konvektionsbereichen 41 placed carrier 36 or a support with fixation / convection areas 42 with gravitationally positive gradients a gap 55 with a gap width of 350 μm to a receiving unit for cell substrate with fixation / convection areas 43 containing a cell substrate 44 , made on the further building a receiving unit 48 with fixing / convection areas for horizontally two radial electrodes 45 that has an insulating spacer and connector 47 are connected, can be placed. Radial electrodes 45 are via vertical holes in fixing bolts of a receiving unit 48 by means of vertical connectors 46 connected. On existing upper bearing surfaces in the receiving unit 48 becomes a further receiving unit for a cell substrate with fixing / convection areas 43 placed through the distance support surfaces to a receiving unit for carriers with Fixier- / Konvektionsbereichen 41 placed carrier with fixation / convection areas 42 with gravitationally negative gradient a gap 55 made with 350 μm and a composite stimulation cell 49 is formed.

In 17 is shown in a chambered lower part 50 a stimulation cell housing (6-well format) for receiving composite stimulation cells 49 a 6-well-format stimulation cell arrangement 51 can be realized with an upper part 52 the Stimulationszellengehäuses with distance ranges for gas exchange and passage openings for connectors 46 can be completed. About the connectors 46 can be any of the six composite stimulation cells 49 via connection lines 60 with the evaluation, storage and control unit 65 (both not shown) in series, in parallel or individually. The areas of the planar, upper and lower beams with fixing / convection areas 42 , the distance ranges for the gas exchange in the upper part 52 as well as the media volumes per well are parameterizable analogous to 6-well standard cultivation systems. This ensures comparability in the conduct of investigations.

According to the invention, the stimulation cell is a non-dynamic, multi-dimensional method for stimulating in vitro cell or tissue growth in a definable interface situation between a tissue-like region and a region in the form of degradable or non-degradable carriers 36 ; in other words, for the stimulation of growth processes in an in situ similar three-dimensional cell or tissue area with opposite boundary layers including a gap 55 ,

The stimulation of cell growth processes in an in vivo / in situ like situation is accomplished by location based generation and temporal variation of electrical parameters of an array of actuators.

LIST OF REFERENCE NUMBERS

1

electrode

1a

electrically insulated electrode

1b

electrically conductive electrode

10

inner space

11

lower part

12

media supply

13

media transfer

14

top

15.1

Blind bore with internal thread

15.2

Through Hole

16

outer electrode

17

border

18

connection

19

magnetic field generator

20

cell substrate

21

Gap for guest exchange

30

cradle

31

fixing

32a

electrically insulated center electrode

32b

electrically conductive center electrode

33

isolation

34.1

magnetic body

34.2

magnetizable body

36

carrier

41

Support unit for carriers with fixation / convection areas

42

Carrier with fixation / convection areas

43

Acquisition unit for cell substrate with fixation / convection areas

44

Cell substrate (for recording unit 43 )

45

radial electrodes

46

connector

47

Spacer and connector

48

recording unit

49

composite stimulation cell

50

chambered lower part

51

Stimulating cell arrangement

52

top

55

gap

60

connecting cable

65

Evaluation, storage and control unit

M

medium

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 3923279 [0002]
• DE 102004054125 A1 [0002]
• DE 3735702 A1 [0004]

Cited non-patent literature

• J Exp Zool. 1990 Feb; 253 (2): 163-76, Harris AK, Pryer NK and Paydarfar D. in "Effects of electric fields on fibroblast contractility and cytoskeleton." [0003]
• ST Curtze (2006, "Tensile Force Microscopy on Osteoblasts", Diss. Human Medicine, University of Marburg: 8 ff. [0003]
• Caubet, R. et al (2004, A radio frequency electric current enhances antibiotic efficacy against bacterial biofilms, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY: 4662-4664) [0006]
• Borden, A. et al. (2007, Prevention of pin tract infection in external stainless steel fixator frames using electric current in a goat model, Biomaterials 28: 2122-2126) [0006]
• Hungerer, S. et al. (2008, Electromagnetic Procedures in Bone Healing Disorders Evidence of this Additive Measure, Trauma Occupational Disease 10 [suppl.2]: 219-225) [0007]
• Schmidt-Rohlfing, B. et al. (2010, Electromagnetic Fields, Electricity and Bone Healing: What Is Secured? Electromagnetic Fields, Electric Current and Bone Healing - What is the Evidence ?, Z Orthop Accident: 1-6) [0007]

Claims (24)

Stimulation cell for in vitro stimulation of cells or tissues, comprising a body enclosing an interior space, comprising an upper part and a lower part and at least one actuator for causing a stimulating effect on at least a portion of the cells or tissue, characterized in that - in the inner space ( 10 ) A carrier ( 36 ), which is colonizable by cells or tissue at least over areas of its surface, and - the actuator is designed so that it can be topically applied to the surface of the carrier ( 36 ) a dosable gradient with a growth of the cells or tissue stimulating effect is evoked. Stimulation cell according to claim 1, characterized in that the carrier ( 36 ) is a molded article of at least one biodegradable material, of at least one non-biodegradable material or of a combination of these materials. Stimulation cell according to claim 1 or 2, characterized in that at least one media feed ( 12 ) and at least one media transfer ( 13 ) on at least one side of the interior ( 10 ) is present for the supply and discharge of a medium (M). Stimulation cell according to one of claims 1 to 3, characterized in that at least two electrodes ( 1 ) are present as an actuator, wherein the electrodes ( 1 ) are designed to generate spatially and temporally defined electrical and / or electromagnetic fields with variable shape, magnitude, direction and intensity. Stimulation cell according to one of the preceding claims, characterized in that the electrodes ( 1 ) from a group comprising electrodes electrically isolated against the medium (M) ( 1a ), against the medium (M) electrically conductive electrodes ( 1b ) and against the medium (M) electrically isolated electrodes ( 1a ) and electrically conductive electrodes ( 1b ) hybrid electrodes are selected. Stimulation cell according to one of the preceding claims, characterized in that the electrodes ( 1 ) are segmented. Stimulation cell according to one of the preceding claims, characterized in that cluster electrodes are present, which consist of mutually grid-shaped electrodes ( 1 ) and / or signal-controlled electrodes ( 1 ) are formed. Stimulation cell according to one of the preceding claims, characterized in that the stimulation cell at least one outside the interior ( 10 ) arranged magnetic field generator ( 19 ) is assigned as an actuator, so that the stimulation cell at least over areas in one by the magnetic field generator ( 19 ) is arranged. Stimulation cell according to one of the preceding claims, characterized in that a hybrid actuator is present, can be caused by the same time or successively different gradients from each other. Stimulation cell according to one of the preceding claims, characterized in that in the interior ( 10 ) at least one receiving device ( 30 ) for receiving at least the carrier ( 36 ) and / or at least one cell substrate ( 20 ) is arranged. Stimulation cell according to claim 10, characterized in that the receiving device ( 30 ) is formed so that between a surface of the in the receiving device ( 30 ) carrier ( 36 ) and a surface of the in the receiving device ( 30 ) cell substrate ( 20 ) A gap ( 55 ) is adjustable, through which the surfaces of the carrier ( 36 ) and the cell substrate ( 20 ) are separated from each other. Stimulation cell according to claim 10 or 11, characterized in that at least one of the receiving devices ( 30 ) a positioning of the carrier ( 36 ) and / or the cell substrate ( 20 ) in the interior ( 10 ), so that in addition to a positioning of the carrier ( 36 ) also a relative movement between carriers ( 36 ) and actuator is effected. Stimulation cell according to one of the preceding claims, characterized in that the carrier ( 36 ) temporarily with at least one magnetic body ( 34.1 ) or a magnetizable body ( 34.2 ) is in contact and this is at least partially in the field lines of a primary magnetic field, so that in the magnetic body ( 34.1 ) or the magnetizable body ( 34.2 ) by the primary magnetic field, a secondary magnetic field is variable. Stimulation cell according to one of the preceding claims, characterized in that the electrodes ( 1 ) are designed as measuring means for measuring electrical quantities. Stimulation cell according to one of the preceding claims, characterized in that the stimulation cell is connected to a controlled drive, by means of which the stimulation cell is controlled to be moved. A method of in vitro stimulation of cells or tissues, comprising the steps of: - introducing a cell substrate carrying the cells or tissue ( 20 ) in an interior ( 10 ) of a stimulation cell; - inserting a carrier ( 36 ), which is settable or already populated at least over settable areas of its surface by the cells or tissue, into a receiving device ( 30 ) in the interior ( 10 ) of the stimulation cell; - introducing a medium (M) for physiological care of the cells or tissues; Producing at least one dosing topical gradient with a cell or tissue stimulating effect on the surface of the carrier ( 36 ). A method according to claim 16, characterized in that measured variables, which are variable as a result of reactions of the cells and tissue to the stimulating effect, are detected and stored. A method according to claim 16 or 17, characterized in that the stimulating effect of certain subregions of the cell substrate ( 20 ) is assigned and limited to this is generated. Method according to one of claims 16 to 18, characterized in that the stimulating effect is selected from the generation of electromagnetic fields, electric fields, magnetic fields, the addition of chemical compounds and the coupling of physical stimuli. Method according to one of Claims 16 to 19, characterized in that initial measured values of at least one measured variable are recorded and stored at an initial measuring time, and the initial measured values are used as reference measured values for measured values acquired at later measuring times. Method according to one of Claims 16 to 20, characterized in that information about the growth of the cells or tissue is derived from the changes in the measured values recorded and stored at different measuring times. Method according to one of Claims 16 to 21, characterized in that the measured values are recorded in a time-resolved and spatially resolved manner. Method according to one of claims 20 to 22, characterized in that control signals for controlling the actuator are generated on the basis of the measured values in order to change the gradient. Method according to one of claims 16 to 23, characterized in that between the cell substrate ( 20 ) and the carrier ( 36 ) A gap ( 55 ) can be adjusted with a gap width.

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