JP2019513360A - Activated platelet composition capable of modulating growth factor levels - Google Patents

Abstract

Methods and systems for producing adjustable or customizable activated production compositions are relevant. In certain embodiments, one or more of the electrical pulse parameters, the flow rate, or the sample container size is altered to produce an activated product composition. The activated product composition can be customized or optimized based on the particular patient or procedure. [Selected figure] Figure 1

Description

  The subject matter disclosed herein relates generally to platelet therapy used in various medical applications such as surgery or treatment of trauma. Certain embodiments relate to enabling platelet activation and growth factor levels to be specified or "modulated" by a user.

  The use of platelet gel (also called "activated platelet rich plasma") can be used in clinics or other medical facilities for various applications including wound healing (eg after surgery) and promoting hemostasis Treatment method. There is interest in the use of platelet therapy as a wound healing treatment, especially for many types of injuries and conditions such as nerve injury, tendinitis, osteoarthritis, myocardial injury, and bone repair and regeneration. Furthermore, the induction of platelet gel used for the patient may be autologous, meaning that platelets are derived from the patient's own tissue and / or body fluid. Thus, a patient's blood sample can be used to obtain a platelet gel used to treat the patient.

  As an example, a doctor can collect blood from a patient. The blood can then be centrifuged to generate platelet rich plasma (PRP). Upon platelet activation, platelets in the blood release growth factors and proteins that facilitate and promote the wound healing cascade. Thus, a clinical workflow may involve taking blood from a patient, centrifuging the blood to separate platelets, and performing in vitro platelet activation such as using bovine thrombin. The activated platelets or platelet gel can then be applied to the wound or other treatment area. If in vivo platelet activation is used instead, the physician can apply PRP to the site without the addition of a platelet activating agent. Platelet activation, including growth factor release and coagulation, is usually induced by collagen in connective tissue.

  In such in vitro applications where thrombin (eg, bovine thrombin) is used to induce platelet activation, the resulting growth factor levels can be fixed based on the biological response. That is, the amount of different growth factors and / or their respective ratios or proportions are determined by the nature of the thrombin-based activation. Thus, in such reactions, the clinician can not adjust or manipulate the respective amounts or proportions of the different growth factors, and instead must use conventional activating compositions.

WO 2015/108778

  Certain specific embodiments commensurate with the scope of the claimed invention at the time of filing are summarized below. These embodiments are not intended to limit the scope of the claimed invention, but merely to provide an overview of the possible forms of the present invention. In fact, the invention can include various forms which may be similar or different to the embodiments described below.

  In one embodiment, a method of releasing a growth factor is provided. According to this method, the sample is positioned relative to the electrodes of the pulse generator. A series of electrical pulse parameters are specified. Different parameter values result in different levels of one or more growths in the activated product composition. The sample is exposed to one or more electrical pulses generated according to the parameter values. The sample, when exposed to one or more electrical pulses, results in an activated product composition having levels of one or more growth factors determined at least partially by the set of electrical pulse parameters.

  In another embodiment, a method is provided for releasing a growth factor. According to this method, the cuvette size is selected. Different cuvette sizes result in different levels of one or more factors in the activated product composition. The sample is placed in a cuvette of a selected size. The cuvette is placed in the sample holder of the pulse generator. The sample is exposed to one or more electrical pulses. When exposed to one or more electrical pulses, the sample produces an activated product composition having levels of one or more growth factors determined at least in part by the cuvette size. The cuvette size can determine the electric field and / or energy density (since the electric field is the voltage divided by the cuvette spacing).

  In a further embodiment, a method is provided for customizing activated blood induced cell treatment. According to this method, a customized growth factor profile for treating a patient is determined based on one or both of the type of wound or the respective process of the wound healing cascade. One or more electrical pulse parameters and cuvette sizes corresponding to the determined growth factor profile are selected. The growth factor profile can be tailored to the particular stage or process of the wound healing cascade. For example, the epithelialization stage may require a growth factor profile in which TGFb1, a growth factor known to promote scarring, is reduced. The granulation stage of wounds may require a growth factor profile that is rich in VEGF, a growth factor known to promote angiogenesis, ie the formation of new blood vessels. The sample placed in the cuvette of the selected size is exposed to one or more electrical pulses generated based on the selected electrical pulse parameters to generate the activated product composition having the growth factor profile. Generate

  In a further embodiment, an electrical pulse generation system is provided. In one embodiment, the system includes a sample holder configured to receive at least two different sized cuvettes, and, when present in the sample holder, to generate one or more electrical pulses to the cuvette. A pulse generating circuit configured, a non-transitory computer readable memory storing one or more user input devices configured to receive user input, and one or more processor executable routines, and computer readable And a processor configured to access and execute one or more processor executable routines stored in memory. The processor executable routine is executed to receive an input specifying one or more electrical pulse parameters corresponding to the prescribed growth factor profile, the different electrical pulse parameter values being growth factor profiles Using pulse generation circuitry to generate an activation producing composition having a prescribed growth factor profile, producing different levels of one or more growth factors at 1 Generating an operation including generating one or more electrical pulses.

  These and other features, aspects, and advantages of the present invention will be better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings. It will be

FIG. 1 is a schematic view of a pulse generation system according to an aspect of the inventive approach FIG. 6 is a graph showing platelet derived growth factor (PDGF) levels in a platelet activation composition produced under various scenarios, in accordance with aspects of the present technique. FIG. 6 is a graph showing epidermal growth factor (EGF) levels in platelet activation compositions produced under various scenarios, in accordance with an aspect of the present technique. FIG. 5 is a graphical representation showing vascular endothelial growth factor (VEGF) levels in platelet activation compositions produced under various scenarios, in accordance with aspects of the present technique. FIG. 6 is a graph showing Transforming Growth Factor Beta 1 (TGFb1) levels in platelet activating compositions produced under various scenarios, in accordance with aspects of the present technique. FIG. 7 shows the results of epithelialization of in vivo cell proliferation assays using growth factors obtained according to some of the present studies. FIG. 5 shows the angiogenesis results of in vivo cell proliferation assays using growth factors obtained according to some of the present studies. FIG. 6 is a process flow diagram showing steps in the generation of a customizable or regulatable platelet activating composition according to an aspect of the present technique.

  In the following, one or more specific embodiments of the present subject matter are described. Efforts to provide a brief description of these embodiments may not result in all features of an actual implementation being described herein. In developing an actual implementation, such as an engineering or design project, many implementation-specific decisions must be made, for example, addressing system-related and business-related constraints, in order to achieve the developer's specific objectives. Also, it should be understood that these constraints may vary from implementation to implementation. Furthermore, although such development work may be complex and time consuming, it is nevertheless understood by those skilled in the art having the benefit of this disclosure that it is a routine task of design, fabrication, and manufacture. I want to be

  When introducing elements of various embodiments of the present invention, the singular expressions “a, an” and “the” mean that there are one or more elements. . The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. It is a thing.

  Platelet activation and / or aggregation may be used to treat wounds in vivo and / or ex vivo. For platelet activation in vivo, inactive platelet rich plasma (PRP) is applied or injected at the site of injury and activated by naturally occurring compounds in the body such as collagen present in connective tissue.

During conventional in vitro processes, platelets in the collected and separated blood are exposed to platelet activating compounds such as thrombin that trigger the release of growth factors (eg, platelet derived growth factor (PDGF)) . For example, for ex vivo platelet activation, a physician can collect blood from a patient and centrifuge the blood sample to produce a platelet rich plasma (PRP) sample. Calcium chloride (CaCl ₂ ) and platelet activating compounds such as thrombin can be added to the PRP sample to induce platelet activation, and then form a gel containing the growth factor to be applied to the wound. However, this process does not allow any way to adjust or regulate the various levels of different growth factors relative to one another. Thus, the clinician must simply use the results of the activation process, regardless of whether the resulting growth factor mixture is optimized for the immediate task.

  Embodiments of the invention discussed herein respond to exposure to one or more customizable energy exposure protocols that allow release of different levels or amounts of growth factors in response to activation parameters. External platelet (or other cell) activation and growth factor release. In addition to the release of growth factors, the approach of the invention can also be used to control the release of other factors in the activation procedure. For example, activated platelets (or other cells in a sample to be exposed) can be regulated or regulated in a manner that is endogenous antioxidants, reactive oxygen species, reactive oxygen species, in response to the procedures discussed herein. Matrix metalloproteinase-2 (MMP-2) and other factors can be released. That is, the adjustable or customizable activation discussed herein can include not only growth factors, but also customized release of other factors that may be associated with the wound healing process. Thus, although specific examples or arguments herein may be provided in the context of growth factors, such examples and arguments also encompass other factors as listed above, and these Also, they can be released in response to different customizable activation protocols.

  The techniques of the present invention can be used with various types of cells that release proteins, growth factors when activated, including but not limited to platelets, red blood cells, white blood cells and the like. In this way, a platelet gel can be generated, where the levels of different growth factors in the platelet gel are modulated, or, for example, to optimize the amount of specific factors generated, or Are adjusted relative to one another to obtain a desired ratio or ratio of certain factors to other factors. This allows for the generation of customized or optimized platelet gels that can have different growth factor profiles, different endogenous antioxidant profiles, different reactive oxygen species profiles, etc. This may be useful because different stages of the healing cascade (eg, angiogenesis, epithelialization, etc.) may be benefited or improved by different growth factors or other factors. Thus, by adjusting growth factor levels based on particular stages of the wound healing process, the wound healing process can be accelerated.

  Ex vivo platelet activation discussed herein elicits platelet activation by exposing a blood sample, such as a PRP sample, or any suspension comprising platelets to an electrical pulse (eg, exposure to a pulsed electric field) However, exposure to other types of energy is also contemplated and encompassed. Methods for ex vivo growth factor release may or may not include the addition of chemicals to the blood sample prior to electrical stimulation. As discussed herein, depending on the parameters of activation exposure, activation may or may not involve destruction (eg, lysis) of cells (eg, red blood cells) in a sample. The process of cell lysis can be adjusted according to the parameters of activation exposure. In certain embodiments, electrical stimulation or activation can be applied using different electrical parameters (eg, amplitude, voltage, electric field, energy density, current, pulse width, number of pulses, etc.) and different parameters or The combination of parameters results in different growth factor levels and / or different proportions of growth factors to each other. Correspondingly, exposure to other types of energy can include the adjustment of one or more parameters that are typically associated with the generation or exposure of that type of energy. Such different formulations of activating compositions may be used to achieve different biological or medical effects (e.g., enhanced wound healing), thus the desired effect may be given to a given cell. The electrical pulse parameters utilized to activate the composition can be determined.

  With the above in mind, FIG. 1 schematically illustrates a pulse generation system 10 for ex vivo platelet (or other cellular product) activation and customizable or adjustable growth factor release. System 10 includes a pulse generation circuit 12 and opposing electrodes (or arrays of electrodes) 14 and 16. In the illustrated embodiment, the electrodes 14 and 16 are spaced apart on opposite sides of the cuvette 18. That is, cuvette 18 is disposed between the electrodes, and electrodes 14 and 16 are coupled to the pulse generation circuit via contacts 20. That is, in the illustrated example, conductive coupling (ie, contact coupling) is shown. However, this example of catalytic coupling is provided only to facilitate the explanation and to provide a useful context to illustrate the approach of the invention, as the sample is to be activated energy (as discussed herein). It should be understood that it is not the only appropriate mechanism for exposure. For example, in other embodiments, non-contact bonding techniques (such as capacitive or inductive bonding techniques) may be used to achieve the discussed energy bonding. Thus, as discussed herein, energy coupling to the platelet suspension involves contact (as shown in this example) between the sample container and the conduit and the electrode through any suitable mechanism. It should be understood that this may occur due to the absence of such contact using inductive or capacitive effects.

  Regardless of the physical or structural embodiment, the pulse generation circuit 12 activates platelets or other cell types within the sample 22 that release proteins and / or growth factors when activated or stimulated during operation. To electrically stimulate or activate the blood, blood component or platelet suspension sample 22 in the cuvette 18. As discussed herein, this is included within the cuvette 18 regardless of the manner in which the electrodes 14 and 16 and the cuvette 18 are physically integrated or interfaced when the pulse generation circuit 12 is operating. It can take the form of applying a pulsed electric field to the sample. In certain embodiments, system 10 may be configured to receive or hold different sized cuvettes, such as cuvettes of different diameters or widths.

  In certain embodiments, the cuvette 18 may be disposable and / or removable from the sample holder 24 that incorporates the electrodes 14 and 16. By inserting the cuvette 18 into the sample holder 24 and bringing the electrodes 14 and 16 into contact with the contacts 20, the pulse generation circuit 12 can generate an electrical pulse across the sample 22. As will be appreciated, cuvette 18 is only one example of a suitable sample container, and is another type of container configured to hold sample 22, contact electrodes 14 and 16, and conduct electrical pulses. , Can be used in combination with the system 10. As discussed herein, the spacing between electrodes 14 and 16 can affect the strength of the electric field of the pulse, which is defined as the ratio of applied voltage to cuvette gap distance. For example, exposure of a 1 cm wide cuvette to a 1 kV pulse results in an electric field strength of 1 kV / cm. The electric field strength, electrode separation distance, and other parameters associated with the generated electrical pulse are varied or adjusted to change growth factor levels relative to one another during the activation procedure, as discussed herein. It is a factor to gain.

  As will be appreciated, the illustrated cuvette or container based activation system is suitable for batch type processing environments. However, a flow-through processing environment may be used instead, and the conduit instead passes through electrodes 14 and 16 which may be on the opposite side of the conduit or surround the conduit. Such a flow through arrangement allows the sample to flow continuously through the conduit to be exposed to a pulsed electric field for activation, and the activation products are collected continuously or semi-continuously. In such embodiments, other parameters may also be adjusted to adjust or customize the activation product, in addition to, or instead of, the electrical parameters at the electrodes and / or the width between electrodes 14 and 16 Good. For example, the flow rate of the sample (eg, platelet suspension) through the conduit and / or the diameter of the conduit may be considered or adjusted as a factor or parameter of the activation process. That is, in addition to the electrical parameters specified for the electrodes, one or both of flow rate and electrode spacing can determine the electric field exposure (or electric field density exposure) to which the sample is subjected during activation.

  In particular embodiments, the system includes control and input circuitry, may be implemented in a dedicated housing, or may be coupled to a computer or other processor based control system. For example, system 10 may include or be in communication with processor 26 controlling pulse generation circuit 12. Additional components of system 10 may include memory 28 for storing instructions to be executed by processor 26. Such instructions may include protocols and / or parameters for generating electrical pulses using pulse generation circuit 12. Processor 26 may include, for example, a general purpose single or multi-chip microprocessor. Further, processor 26 may be any conventional dedicated processor, such as an application specific processor or circuit. Memory 28 may be any suitable non-transitory computer readable medium, such as a random access memory, a mass storage device, a solid state memory device, or a removable memory. Additionally, display 30 can provide instructions to an operator associated with the operation of system 10. The system 10 activates the pulse generation circuit 12 to select or specify appropriate pulse parameters, or is preconfigured from among a number of such profiles (such as profiles corresponding respectively to different stages of different wound healing). A user input device 32 (eg, a keyboard, a mouse, a touch screen, a trackball, a handheld device such as a PDA or smart phone, or any combination thereof) may be included to select the pulse profile.

  The pulse generation system 10 discussed herein is exposed to electrical stimulation as a single purpose device for platelet or other cell type activation, or in addition to electroporation, platelet (or other cell type) activation. May be implemented as a multipurpose device that may be used for other electric field exposure applications such as promoting cell growth via. Further, system 10 may be modified to generate different levels or proportions of growth factors according to one or more defined protocols and / or as discussed herein. The plurality of parameters may be configured to generate an electrical pulse. The protocol may be generated by user input and / or stored in memory 28 for selection by the user, such as from a list or menu. In one embodiment, the pulse generation circuit 12 uses one or more growth factor levels using a specific field strength, pulse length, total exposure time, flow rate (in a flow-through embodiment) or other characteristics. In order to implement a protocol to generate a customized activated cell composition (eg, a gel customized to enhance a particular phase of wound healing) determined by designated pulse parameter values, It can operate under control. Such protocols may be empirical or theoretical, such as to account for desired biological or medical effects (eg, one step of wound healing) and / or destruction or lysis of cells of the sample during activation. Can be determined by In other embodiments, system 10 may be configured to receive user input associated with one or more of field strength, pulse length, flow rate, and / or total exposure time, ie, the user , One or more of these operating parameters can be changed or specified. Further, system 10 may be configured to generate a series of pulses that may differ from one another to generate a particular pulse shape or according to user input and / or stored protocol settings.

  By way of example, in one embodiment, the pulses generated by system 10 may have a duration of about 1 nanosecond to about 100 microseconds and an electric field strength of about 0.1 kV / cm to about 350 kV / cm, depending on the application. You can have As mentioned above, the electric field strength of the pulse is the applied voltage divided by the distance between the electrodes 14 and 16. The pulses generated by system 10 typically have an electric field strength of 0.1 kV / cm or more, but the pulses do not typically exceed the breakdown electric field of the suspension containing the cells.

  In some embodiments, pulse generation system 10 can include a sensing function. That is, the pulse generation system 10 can be configured to expose the sample 22 to a detection signal, which can be an electrical pulse having an electric field strength lower than that of the electric pulse used for cell activation. The pulse generation system 10, as shown in FIG. 1, acquires and / or processes sensing signals to estimate some of the electrical properties of the sample 22, including but not limited to conductivity and permittivity. Can include a current sensing circuit 34 that can. The current sensing circuit 34 may be coupled to a processor 26 that may control the generation and processing of the sensing signal, and may, in some embodiments, perform some of the processing. In other embodiments, current sensing circuit 34 includes a dedicated processor that controls the processing of the sensing signal and can be in communication with processor 26 to report results. Alternatively, the current sensing circuit 34 may be integral with the pulse generation circuit 12 to provide an input used to generate the next activation electrical pulse. In still other embodiments, processing of the sensing signal may be performed by a dedicated processor or processor 26 as described above.

For various electrical pulsing factors or parameters, these factors are not limited, but cuvette spacing (i.e., the width of the cuvette 18 to which the pulse is applied), flow rate (in flow-through embodiments), voltage, electric field (in flow-through embodiments) For example, intensity or density), current, pulse width, and the number of pulses applied. In one study, these are related to other controls or activation scenarios (eg, untreated platelet rich plasma (PRP), PRP + calcium chloride (CaCl ₂ ), PRP + thrombin (eg, bovine thrombin activated PRP)). The combination of parameters of was tested. Table 1 summarizes the various combinations of electrical pulse parameters used in studies to activate platelets using electrical stimulation.

  Additionally, the last column of Table 1 indicates whether hemolysis occurred in the sample when exposed to pulses having the listed parameters. The electrical parameters can be adjusted to avoid or not avoid hemolysis, ie erythrocyte lysis. In the illustrated example, hemolysis in the sample was observed in the two scenarios where the electric field and current are highest. Thus, among other considerations, the fact that cell lysis or disruption in the sample is desirable or undesirable is a consideration in selecting electrical or other parameters (eg, electrode spacing and flow rate) for the activation protocol. It can be a matter.

The results of this study are shown in FIGS. FIG. 2 graphically illustrates the measured platelet derived growth factor (PDGF), FIG. 3 graphically illustrates the measured epidermal growth factor (EGF), and FIG. 4 illustrates the measured vascular endothelial cell growth factor (EGF). FIG. 5 graphically depicts VEGF) and FIG. 5 graphically depicts measured transforming growth factor beta (β) 1 (TGFb1). As shown in these results, differently parameterized electrical pulses are not only between electrical stimulation results and non-electrical stimulation results, but also during differently parameterized electrical stimulation scenarios, respectively. Brought about different levels of growth factor. In these figures, the experimental conditions are: PRP = platelet rich plasma (not activated), calcium = platelet rich plasma + calcium chloride, thrombin = platelet rich plasma + calcium chloride + bovine thrombin (platelet activating factor), 1 , 2, 3, 4, 5 = platelet rich plasma + calcium chloride + activation by electrical stimulation conditions 1 to 5 shown in Table 1.

  As an example, the electrical pulse and cuvette spacing parameterized as shown in Figure 1 in Table 1 in connection with PDGF and as shown in Figure 2 are four other scenarios (approximately 8,000 pg PDGF / PDGF levels (about 3) which are different from 3 mL non-electrical scenarios (about 600 pg PDGF / mL for PRP, about 6,000 pg PDGF / mL for calcium, about 8,000 pg PDGF / mL for thrombin) , 800 pg PDGF / mL). Thus, by manipulation of any combination of electrical and spatial factors such as electrical pulse characteristics, cuvette spacing or width, and / or energy density found in the sample during the pulse, different levels of within the activated platelet composition PDGF was obtained.

  Similarly, electrical pulses and cuvette intervals parameterized as shown in scenarios 1 to 5 of Table 1 in relation to EGF and as shown in FIG. 3 are scenarios for each numbered scenario This resulted in considerably different levels of EGF ranging from about 100 pg EGF / mL of 1 to about 3,300 pg EGF / mL of scenario 3. Furthermore, the level of EGF measured for thrombin activated platelets was about 500 pg EGF / mL, which was the highest level observed for non-electrically activated samples. Thus, by manipulation of any combination of electrical and spatial factors such as electrical pulse characteristics, cuvette spacing or width, and / or energy density found in the sample during the pulse, different levels of within the activated platelet composition EGF was obtained.

  For VEGF (FIG. 4), different electrical pulse parameters and cuvette spacing as shown in scenarios 1 to 5 of Table 1, scenario 1 and scenario 2 result in approximately 800 pg VEGF / mL and approximately 1,750 pg VEGF / mL, respectively. A similar result is seen in that scenario 3-5 resulted in different levels of VEGF for each numbered scenario, resulting in between about 2,250 pg VEGF / mL and about 2,500 pg VEGF / mL. Be Thus, as in the previous embodiments, activation is accomplished by manipulation of any combination of electrical and spatial factors such as electrical pulse characteristics, cuvette spacing or width, and / or energy density found in the sample during the pulse. Different levels of VEGF within the rendered platelet composition were obtained.

  Finally, the electrical pulse and cuvette intervals parameterized as shown in scenarios 1 to 5 of Table 1 in relation to TGFb1 and as shown in FIG. 5 are scenarios for each numbered scenario The range from 1000pg TGFbl / mL of 2 to approximately 65,000pg TGFbl / mL of scenario 3 and other scenarios resulted in significantly different levels of TGFbl that fall between these values. Thus, as in the previous embodiments, activation is accomplished by manipulation of any combination of electrical and spatial factors such as electrical pulse characteristics, cuvette spacing or width, and / or energy density found in the sample during the pulse. Different levels of TGFb1 within the reconstituted platelet composition were obtained. As a further example, electrical condition 2 produces lower levels of TGFb1 compared to thrombin activation (FIG. 5) but with the same levels of PDGF-aa (FIG. 2), VEGF (FIG. 4) and thrombin. Generate EGF (Figure 3). Since the scientific literature suggests that TGFb1 correlates with increased scarring, the platelet gel produced by electrical stimulation 2 described herein is compared to platelet gel produced by thrombin activation. And may induce lower scarring.

  From the above examples it can be seen that different growth factors are released differently depending on various different aspects of the electrical pulse and / or electrode spacing. Thus, as appreciated, the electrical pulse or pulse sequence can be parameterized to release the desired growth factor level based on the desired growth factor profile. In addition, pulse parameters can be varied or adjusted between pulses to target different growth factor emissions by different pulses.

  It is also noteworthy that spatial variations due to electrode spacing appear to be responsible for sometimes different growth factor emissions. For example, scenarios 2, 3 and 4 are all performed with a cuvette spacing of 2 mm, but at least in the context of EGF, VEGF and TGFb1 release, each different electrical pulse parameter will be released as a respective growth factor scenario The result of 4 was larger than that of scenario 2 and the result of scenario 3 was larger than that of scenario 4. That is, in these scenarios, the cuvette spacing remains constant, so the different factor is the difference in electrical pulse parameters.

  Conversely, for Scenario 2 and Scenario 5, roughly similar electrical pulse parameters were applied for electric field, current, pulse width, and number of pulses, but the cuvette spacing was different (2 mm for Scenario 2 and Scenario 5) 4 mm). In this case, different amounts of EGF, VEGF and (especially) TGFb1 are released in these two scenarios, which result in different cuvette spacing or some derived parameter of cuvette spacing (such as energy density or electric field) It strongly suggests that it is a factor. Thus, both electrical pulse parameters and spatial factors (separately or in a mixed manner), in an absolute sense and / or relative to other growth factors, of one or more growth factors It may affect (e.g., preferentially) the release differently.

  Thus, using this knowledge, with one or more specific growth (or other) factors of a desired amount, one or more factors with a desired ratio or ratio to other factors, or It is possible to produce platelet activating compositions (eg, gels) having factors of a particular profile in an absolute or relative sense. That is, platelet-activating compositions can be used by clinicians such as compositions specific to certain stages of wound healing (eg, angiogenesis, ie, higher VEGF for wounds requiring new blood vessels, etc.) With the appropriate combination of electrical pulse parameters, electrode spacing, and / or flow rates, which allow to produce or order a platelet activating composition having the desired medical or biological effect for a given patient It can be adjusted or customized. In practice, this may be implemented using preset options programmed into the system 10, such as selectable buttons or items of a menu or list of a graphical interface, each option having a different medical effect or prescription Or, different sets of pre-configured electrical pulse parameters, thus corresponding to different growth factor profiles of the composition. However, even in such a configuration, the user can be provided with the option to enter custom or user specified electrical pulse parameters.

  As a further example, FIGS. 6 and 7 show in vivo cell proliferation assays using inactive PRP, bovine thrombin activated PRP, and growth factors from electrical conditions 2, 3 and 5 from Table 1. The results are shown (see growth factor profiles in FIG. 2, FIG. 3, FIG. 4 and FIG. 5). These assays used human dermal fibroblasts (FIG. 6) as a surrogate for epithelialization (FIG. 6) and human dermal microvascular endothelial cells as a surrogate for angiogenesis (FIG. 7). FIG. 6 shows that electrical conditions 2, 3 and 5 induce cell proliferation at a higher rate compared to thrombin activation (faster epithelialisation). However, FIG. 7 shows that electrical state 3 induces a higher growth rate (faster angiogenesis) compared to bovine thrombin activation, and electrical conditions 2 and 5 grow slower (slow compared to bovine thrombin). Angiogenesis) is shown. These assays demonstrate the possibility of allowing clinicians to modulate wound healing effects with appropriate electrical stimulation of platelet rich plasma.

  With the above in mind, FIG. 8 shows an example of a process flow 40 that may be used to generate a platelet activating composition 50 as discussed herein. Certain steps of the method 40 may be performed by an operator while other steps of the method are performed by the system 10, such as executing one or more algorithms that control the configuration and / or application of electrical pulses. Understand that you can do it.

Referring to FIG. 8, in one embodiment, a blood sample 42 (eg, a whole blood sample) is first processed to generate a PRP sample 46 (step 44). In an autologous embodiment, the blood sample 42 can be obtained from the patient himself, either during the previous visit or during the current treatment session. In other embodiments, the blood sample may be obtained from a person other than the patient. Furthermore, it is understood that the illustrated process step 44 may not be present in some instances, and that the subsequent steps are instead performed on the blood sample 42. When performed, process step 44 may be one or more techniques suitable for platelet separation such as centrifugation or filtration, or any other suitable technique for producing PRP sample 46 from blood sample 42. It can be based. In certain embodiments, CaCl ₂ can be added to the sample (whether PRP or whole blood) prior to being exposed to one or more pulses at step 52 via the system 10.

  The illustrated process flow shows a step 48 in which the operator selects a cuvette or conduit size (e.g., electrode spacing) in which the sample (whether PRP or whole blood) is placed while being exposed to electrical pulses. ing. Any suitable electrode spacing (eg, 2 mm, 4 mm, or other suitable size) can be employed, and as noted above, the choice of cuvette size has a relative meaning (ie, other growth factors) The level of one or more growth factors present in the activated platelet composition, either proportionally or in an absolute sense (ie, the total amount or concentration present, independently of other growth factor levels) It can be a factor of

  The PRP or blood sample in the selected cuvette size is placed in system 10 and is ready to be exposed to one or more electrical pulses at step 52. Prior to pulse generation, pulse parameters 54 may be selected by the operator or otherwise specified. Depending on the embodiment, any electrical characteristics of the pulse (including but not limited to voltage, electric field, current, etc.), and pulse width (ie duration) and number of pulses applied are directly: Alternatively, corresponding to the activated platelet composition 50 being formulated, such as composition 50 optimized or customized to provide a specific biological or medical effect when used (step 60) It can be specified at step 56 by the operator by selection of the established protocol (e.g. from a menu or list of protocols).

  Although platelet activation is referred to in certain of the present examples, other cell types (eg, red blood cells, white blood cells, etc.) that may also be present in platelet rich plasma may be proteins and / or described herein. It can be activated to release growth factors. That is, the method of the present invention allows proteins and / or growth factors derived from various types of cells, as well as platelets, to be customizablely released when such cells are activated. In general, it can be understood. Furthermore, as mentioned above, while growth factors are mentioned as an example in certain parts of the above discussion, other factors such as endogenous antioxidants, reactive oxygen species, matrix metalloproteinase 2 (MMP-2 ) May be present in the activated product and may have customized or adjusted levels or proportions as discussed herein.

  One or more of the disclosed embodiments, alone or in combination, have one or more technical effects useful for medical techniques for ex vivo platelet activation and release of growth factors and other factors. Can be provided. The present technique for platelet activation in vitro relates to the amount or concentration of one or more growth factors or other factors, or relative ratios of one or more factors to other factors in the composition. Using cuvettes or conduits of different sizes and / or electrical pulses with different electrical characteristics, durations and / or different numbers of such pulses to customize or adjust the activated platelet composition to enable. In this way, growth factor levels different from those observed when activation is achieved by other means, including chemical means (eg, by exposure to thrombin, collagen, calcium, etc.) An activated platelet composition is produced. The technical effects and technical problems described herein are provided by way of example only and are not intended to be limiting. It should be noted that the embodiments described herein may have other technical effects and may solve other technical problems.

  While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and variations as fall within the true spirit of the invention. Some embodiments can be used for in vivo platelet activation workflows. Without clotting, electrical stimulation can induce growth factor release in PRP and this PRP can be injected at the site of injury. The growth factors thus released can be used for wound healing at the site of injury. Furthermore, in certain embodiments, platelets can also be fully activated by collagen in connective tissue.

DESCRIPTION OF SYMBOLS 10 pulse generation system 12 pulse generation circuit 14 electrode 16 electrode 18 cuvette 20 contact 22 platelet suspension sample 24 sample holder 26 processor 28 memory 30 display 32 user input device 34 current detection circuit 40 process flow 42 blood sample 46 PRP sample 50 activity Platelet composition, platelet activating composition 54 pulse parameters

Claims (20)

A method for releasing growth factors,
Positioning the sample (22) relative to the electrodes (14, 16) of the pulse generator;
Specifying a set of electrical pulse parameters (54), wherein different parameter values result in different levels of one or more growth factors in the activation product composition (50);
Exposing the sample to one or more electrical pulses generated according to the parameter value, the sample (22) being exposed to the one or more electrical pulses, the electrical pulse parameters Providing the activated product composition (50) having a level of the one or more growth factors determined at least in part by a set of.   The method of claim 1, wherein the sample (22) comprises one of a platelet rich plasma sample, a platelet suspension, or a whole blood sample. Selecting a container or conduit size from a plurality of container sizes;
Placing the sample (22) in a container of the preselected size, positioning the sample between the electrodes (14, 16) with the container holding the sample Placing the activated product composition (50) at one or more levels at least partially determined by the container size. Method 1 described.   Positioning the sample (22) between the electrodes (14, 16) comprises flowing the sample through a conduit between the electrodes (14, 16), the activation product composition (50) The method according to claim 1, wherein the one or more factors have a level determined at least partially by one or both of the diameter of the conduit or the flow rate of the sample (22) through the conduit.   The method of claim 1, wherein the set of electrical pulse parameters (54) comprises one or more of voltage, electric field, current, pulse width, energy density, energy per platelet, or number of pulses.   The one or more factors include one or more of platelet derived growth factor, epidermal growth factor, vascular endothelial growth factor, or transforming growth factor beta 1, platelets, red blood cells or white blood cells The method of claim 1, comprising a growth factor released by one or more.   The method of claim 1, wherein cells of the sample (22) are not lysed in response to the one or more electrical pulses. A method for releasing growth factors,
Selecting a cuvette size, wherein different cuvette sizes result in different levels of one or more growth factors in the activated product composition (50);
Placing a sample (22) in the selected size cuvette (18);
Placing the cuvette (18) in a sample holder (24) of a pulse generator;
Exposing the sample (22) to one or more electrical pulses, the sample (22) being at least partially dependent on the cuvette size when exposed to the one or more electrical pulses Providing the activated product composition (50) having the determined level of the one or more growth factors.   Specifying the set of electrical pulse parameters (54), wherein different parameter values result in different levels of one or more growth factors in the activation producing composition (50), the activation producing composition The method according to claim 8, wherein an entity (50) has the one or more growth factors at a level determined at least partially by the set of electrical pulse parameters (54).   10. The method of claim 9, wherein the set of electrical pulse parameters (54) comprises one or more of voltage, electric field, current, pulse width, energy density, or number of pulses.   9. The method of claim 8, wherein the one or more growth factors comprise one or more of platelet derived growth factor, epidermal growth factor, vascular endothelial growth factor, or transforming growth factor beta 1.   9. The method of claim 8, wherein cells of the sample (22) are not lysed in response to the one or more electrical pulses. A method for customizing activated blood induced cell treatment, comprising:
Determining a customized growth factor profile for treating the patient based on one or both of the type of wound or the respective process of the wound healing cascade;
Selecting one or more electrical pulse parameters (54) and cuvette sizes corresponding to the determined growth factor profile;
The grown sample is exposed to the one or more electrical pulses generated based on the selected electrical pulse parameters (54) in the selected size cuvette (18). Generating an activated product composition (50) having a factor profile.   The step of determining the growth factor profile comprises the steps of: platelet-derived growth factor, epidermal growth factor, vascular endothelial growth factor, or transforming growth factor beta 1 to be present in the activated platelet composition (50) 14. The method of claim 13, comprising determining the absolute amount of growth factor released from one or more of platelets, red blood cells or white blood cells, including one or more.   The step of determining the growth factor profile comprises: platelet-derived growth factor, epidermal growth factor, vascular endothelial cell growth factor, or transformed growth to be present in the activation producing composition (50) for another growth factor 15. The method of claim 13, comprising determining the relative amount of one or more of factor beta 1.   The method according to claim 13, wherein selecting the electrical pulse parameter (54) comprises designating one or more values of voltage, electric field, current, pulse width, energy density or number of pulses. Method.   The method of claim 13, wherein selecting the one or more electrical pulse parameters (54) comprises selecting a preconfigured pulse profile from a plurality of preconfigured pulse profiles. An electrical pulse generation system (10),
A sample holder (24) configured to receive at least two different sized cuvettes (18);
A pulse generation circuit (12) configured to generate one or more electrical pulses in the cuvette (18) when present in the sample holder (24);
One or more user input devices (32) configured to receive user input;
Non-transitory computer readable memory (28) storing one or more processor executable routines;
A processor (26) configured to access and execute the one or more processor executable routines stored in the computer readable memory (28), the processor executable routines being executed And receiving an input specifying one or more electrical pulse parameters (54) corresponding to the prescribed growth factor profile, wherein different electrical pulse parameter values are at different levels of one of said growth factor profile. Receiving, providing one or more growth factors; and using the pulse generation circuit (12) to generate an activated product composition (50) having the prescribed growth factor profile. Generating one or more electrical pulses based on the To As, the electrical pulse generating system (10).   The system (10) of claim 18, wherein receiving the input comprises receiving a user selection of the growth factor profile from a plurality of growth factor profiles.   The system (10) according to claim 18, wherein the one or more electrical pulse parameters (54) include voltage, electric field, current, pulse width, energy density, or number of pulses.