CA3094496A1 - Automatic additive multi stage portable three dimensional device for manufacturing of hard and soft organs - Google Patents

Abstract

The use of bio-printers in regenerative medicine used in recent years for these purposes. The basic problems of these inventions are the lack of mechanical strength in the absence of complete processes for the injection of different materials with distinct cells, the lack of stabilization of the biological substance (bio ink) and low uniform cell distribution. The solution is production of a new portable augmentative device incorporates precise control mechanisms with the isolated chamber with hybrid bioprinting system for the production of soft and hard tissue with the morphology of the organ. With structural modification mechanism of the low power laser an plasma also incresea compatibility of polymers and increasing cell adhesion and integration in the scaffolds.

Description

Description Title of Invention: Automatic additive multi stage portable three di-mensional device for manufacturing of hard and soft organs [0001] This application relies on disclosure of and claims priority to and benefit of filing date of Islamic Republic of Iran Provisional application No.
139650140003014980, filed 18 ,March, 2018, the disclosure of which is hereby incorporated by reference herein in its entirety

[0002] This Invention relates to a bio printing field for cloning hard and soft organs and scaffold surface treatment .This invention also relates to methods of using different nozzles and actuators to construct special shaped organs.
Background Art

[0003] Tissue engineering and regenerative medicine is implementing for alleviating of organ shortages to improve patient's serious conditions. The majority of current bio printers are using bio ink such as hydrogel with cells in three dimensional shapes to direct cells to for growth and integrations to special tissue. These systems are using 3 degree freedom additive manufacturing systems technologies by printing layer by layer. There are several restriction for these technologies such as precise multiple cell placement, low density cell purity ,less cell interaction, prolonged material degradation for bio film, scaffold toxicity , separation of cells in one porous scaffold, separated medium transfer for specific cells for differentiation.

[0004] ] Scaffold surface treatment less cell interaction, scaffold toxicity, separation of cells in one porous scaffold is remain a significant challenge

[0005] Bio printer chamber, limited time of printing due to cell vitality there is still a further need to concur these defects.

[0006] There is a need for hybrid bio printer that contain different nozzles and also different actuators for printing different materials and cells. This system also could cross link, making polymerization and have important role in surface treatment during printing process is important factor to clone hard and soft organ together same as structure of human body organs.
Summary of Invention

[0007] Currently most of conventional bioprinters are widely implement in regenerative medicine and tissue engineering applications. Most of these bioprinter are using hydro gels with cells and they are manufacturing scaffolds with three dimensional layer by layer. This innovation with state of art technology able to clone detail required to reca-pitulate hard and soft tissue for human organ simultaneously. It could use several materials such as hydrogels, polymers, liquid composite, drugs and special cell media with unique intelligent injection system. With intelligent control system it could control the printing parameter automatically and its unique chamber is with incubation parameters so the printing time could be added and the patient own stem cells vitality is assured. The unique technique for separation and isolation of different stem cells with nano fiber scaffold is developed by electro- spinning nano fiber texture scaffold printing system. Highly customized and physiologically relevant 3D human tissues form patient own CBCT, MRI and other DCOM files is converted and edited and the sophisticated printing process were done by MOC software and the final sophisticated organ will print.
Technical Problem

[0008] The widening gap between the demand and the supply of organ transplants represent a substantial unmet need amongst the patients and doctors. The current manufacturing capabilities are not adequate to produce artificial organs with precise shape and size of miniature organ parts with no hard and soft organ functional lack of concurrent technology for injection and efficient usage of this technology. Lack of Scaffold Surface treatment, Incubator chamber, limitation of hybrid material injection systems and separation of each tissue cells and conduct media in microfluidic tunnels pattern as another restrictions.
Solution to Problem

[0009] Described herein are a bioprinter with four degree of freedom with four printing head and isolated chamber with support assembly and the controller for sequence of printing and condition monitoring. The controller is configured with three methods via computer, manually by panel and with Wi-Fi feature thro Mobile application.
During the disposition of injection materials such as hydro gel, bio polymer, composite with four external infusions injection pumps each layer of tissue would be fabricated. The placement of multiple cell type in porous construct, each type of cells could be isolated via electro spinning fibred scaffold and the microfluidic tunnels could be specifically designed to reach the specific media to these cells for cell maturation. The injection nozzles assembled in rotated disc is receiving raw materials such as bio inks and bio polymers filament and dispense them according to printing pattern.

[0010] Optionally, Four other actuator and nozzles such as low power laser, low plasma arc, Electro spinning nozzle and composite nozzle could be added to disc.

[0011] Electro spinning system is used for nano fiber based scaffold generation system.
100121 Optionally, low powers laser irradiator with 800nm wavelet actuator for surface treatment for decrease cell adhesion and vitality and also increase proliferation.
[0013] Optionally low Plasma arc deposition actuator for scaffold hydrophobic purpose is used to isolate each tissue segment and covey the media for special cells for maturation and differentiation.
[0014] Optionally, the nozzle assembly printer head have coaxial mechanism so it have inner and outer chamber to inject fluid with drug and gels components with cells si-multaneously.
[0015] Optionally By controlling injection chamber same as co2 incubator parameters for cell growth and expression, parameters such as set temperature body is set to be 37 degree centigrade, CO2 gas serve to maintain pH to natural 7.4, similar to tension in blood stream, high humidity to evaporation of media of (85-95%), for proper culturing and increase cell vitality in long process of printing. To control the temperature the controller set s and the heater elements and fan could control heat by heat transfer process and thermal sensor is used in close loop control system. For controlling Co2(0-20%) and air(02-N2) the air injection pump with controlling output flow of Co2 and air, were used to maintain the parameters related to set points.
Advantageous Effects of Invention [0016] Most of bio 3D printers are using three-axis displacement mechanism, and a single-Duo nozzle pump. This invention have the advantage of using the fourth axis of the rotary substitute disk to use different Actuators, nozzles, coaxial nozzles, lasers, and plasma simultaneously . This is completely different from the design of the mechanism in terms of the design and structure of the degree of freedom. Scheme mechanism is used in axes x, y, z, and the fourth axis is connected directly to the motor with a coupling, which follows a completely different structure and goals. The use of coaxial nozzles instead of a single-pump nozzle can be used to inoculate the drug, as well as the injection of hydrogels like alginate and calcium chloride injections to fix and cross-link the hydrogel with controlling UV, heat condition.
[0017] In the preceding inventions, the use of a single or multiple nozzles submersible pump is printing in a single unit inside the container, but the four injection pumps of this invention are located outside the of the body bio printer. In order to inject bio-polymeric materials, electrically, hydrogels and cells and composites by tube Connected with nozzles. Differences and Advantages of this innovation is it does not require the opening and closing of the enclosure door to replace the injected fluid and control the parameters of the external environment in the operator's function and ob-servation with long printing time, which is an innovative application of this statement.
[0018] Another advantage of this invention is that in general there is no mechanism for modifying the structure with low powered plasma and laser diffusion surface treatment during printing. This feature would increase cell vitality and integrations.
[0019] In the preceding inventions, the use of a single or multiple nozzles submersible pump is placed in a single unit inside the container, but the four injection pumps of this invention are located outside the outside of the body. In order to inject bio-polymeric materials, electrically, hydrogels and cells and composites by tube Connected with nozzles. Differences and Advantages This innovation does not require the opening and closing of the enclosure door to replace the injected fluid and control the parameters of the external environment in the operator's function and observation, which is an in-novative application of this statement. Using the mechanism of external quad pumping pumps can change the contents of the injection, as well as control the temperature and integrity and homogeneity of the injected fluid without opening and closing the printer.
This will not stop the printing and penetration of the organs during the process, and the mechanism. The combination of hard and soft organs, due to the isolation of scaffolds by electrolysis, has created the differentiation of stem cells, and also the pathway for media and growth factors in bio-reactors to printed organs is possible, which is one of the advantages and advantages of this multi-stage printer.
[0020] In the previous invention, the one single-layer body was integrated with an integrated chamber, is provided. One of the advantages of the invention come from a double-glazed body, the use of composite and ABS polymer in internal and external structures with an internal strengthening shell to increase the structural stability and create The closed environment is designed to create an incubator environment inside the chamber and has been designed and manufactured in a very distinct and more functional form with a more static and ergonomic design for better user access.
[0021] The two instantaneous coolant control items are intended, and the invention of the gradual cooling mechanism is used. Another benefit of the incubation environment is that the control of the conditions of the cell culture media is adjusted to the input according to the culture conditions of each cellular tissue. To control the temperature, three heat elements are installed in the device, one on the bottom of chamber on in heat sink on middle, and one on the top of extruder. which creates heat. Both the one is mounted on the extruder of the filament and one on the back of the device, which is different in terms of the mechanism of transmission and temperature reduction.

Creating an incubator environment inside the printer chamber increases the print time from 30 minutes due to the death of stem cells for several hours.
[0022] Most of bio 3D printers are using three-axis displacement mechanism, and a single-Duo nozzle pump. This invention have the advantage of using the fourth axis of the rotary substitute disk to use different Actuators, nozzles, coaxial nozzles, lasers, and plasma simultaneously . This is completely different from the design of the mechanism in terms of the design and structure of the degree of freedom. Scheme mechanism is used in axes x, y, z, and the fourth axis is connected directly to the motor with a coupling, which follows a completely different structure and goals. The use of coaxial nozzles instead of a single-pump nozzle can be used to inoculate the drug, as well as the injection of hydrogels like alginate and calcium chloride injections to fix and cross-link the hydrogel with controlling UV, heat condition.
[0023] In the preceding inventions, the use of a single or multiple nozzles submersible pump is printing in a single unit inside the container, but the four injection pumps of this invention are located outside the of the body bio printer. In order to inject bio-polymeric materials, electrically, hydrogels and cells and composites by tube Connected with nozzles. Differences and Advantages of this innovation is it does not require the opening and closing of the enclosure door to replace the injected fluid and control the parameters of the external environment in the operator's function and ob-servation with long printing time, which is an innovative application of this statement.
[0024] Another advantage of this invention is that in general there is no mechanism for modifying the structure with low powered plasma and laser diffusion surface treatment during printing. This feature would increase cell vitality and integrations.
[0025] In the preceding inventions, the use of a single or multiple nozzles submersible pump is placed in a single unit inside the container, but the four injection pumps of this invention are located outside the outside of the body. In order to inject bio-polymeric materials, electrically, hydrogels and cells and composites by tube Connected with nozzles. Differences and Advantages This innovation does not require the opening and closing of the enclosure door to replace the injected fluid and control the parameters of the external environment in the operator's function and observation, which is an in-novative application of this statement. Using the mechanism of external quad pumping pumps can change the contents of the injection, as well as control the temperature and integrity and homogeneity of the injected fluid without opening and closing the printer.
This will not stop the printing and penetration of the organs during the process, and the mechanism. The combination of hard and soft organs, due to the isolation of scaffolds by electrolysis, has created the differentiation of stem cells, and also the pathway for media and growth factors in bio-reactors to printed organs is possible, which is one of the advantages and advantages of this multi-stage printer.
[0026] In the previous invention, the one single-layer body was integrated with an integrated chamber, is provided. One of the advantages of the invention come from a double-glazed body, the use of composite and ABS polymer in internal and external structures with an internal strengthening shell to increase the structural stability and create The closed environment is designed to create an incubator environment inside the chamber and has been designed and manufactured in a very distinct and more functional form with a more static and ergonomic design for better user access.
[0027] The two instantaneous coolant control items are intended, and the invention of the gradual cooling mechanism is used. Another benefit of the incubation environment is that the control of the conditions of the cell culture media is adjusted to the input according to the culture conditions of each cellular tissue. To control the temperature, three heat elements are installed in the device, one on the bottom of chamber on in heat sink on middle, and one on the top of extruder, which creates heat. Both the one is mounted on the extruder of the filament and one on the back of the device, which is different in terms of the mechanism of transmission and temperature reduction.

Creating an incubator environment inside the printer chamber increases the print time from 30 minutes due to the death of stem cells for several hours.
Description of Embodiments [0028] Specific aspects of this invention consist of two distinct but related aspect 1 and 2.
Aspect 1 is the bio-printer mechanism and the aspect 2 is injection pump mechanism.
[0029] Aspect 1, bio printer mechanism comprising :four degree of freedom three linear movement with lead and screw mechanism coupled to stepper motor and containing rotating disk that connected to stepper motor ,double layer printing chamber with in-cubation parameters , nozzles ,extruder ,Actuators and rotating disk.
[0030] According to aspect 1 Injection of the polymer, the hydrogel components with cells, bio composite and drug will be controlled by the velocity injection pump mechanism.
These hydrogel injection nozzles are pumped cells and chemical crosslinking sta-bilizing solution.
[0031] According to aspect 1 for injection of hydrogels, nano particles and drugs, a variety of polymer solutions and various types of fluids such as media, growth factors, sta-bilizers, other factors could be used. simple four-injection pumps mechanism is used to increase the rate, pressure and flow rate with regard to the sequence of printing and type of density of materials for printing.
[0032] Aspect 2 is injection pumps of any of aspects 22-28 , wherein there is positive pump pressure inside housing witch is in range 1 -20 kPa.
[0033] According to aspect 2 All movements are powered by four 1.8-degree, 4.5 watt stepper motors, with a total accuracy of 0.00027 mm that can be moved around all axes. The main controller of the device, based on the definition process, gives the commands to the motors for the displacement of the plates (three axes) or the rotary transmission mechanism to change the injection and correction mechanisms of the Disk and plate.
[0034] According to aspect 3 which involves the extrude the bio gradable polymers scaffold with the filament extraction mechanism (biodegradable polymers such as PVA,PLGA-PLA-PLLA,PCL,PETG). The temperature and speed of the polymer injection are adjusted according to the type of polymer of the filament. The best result was obtained with a nozzle of 0.2 and 0.1 and a temperature of about 40 to 50 degrees Centigrade. The temperature of the element and the environment was reduced by the fan at top of extruder.
[0035] According to 4 the Fan with nano filter is used to decrease the temperature of chamber and it could operate from 0 to 100 percent performance that could be controlled by operator from computer program.
[0036] According to aspect 5 the filament injection mechanism act as extruder it dragged and moved by the stepper motor. Motor Stepping angel and its movement are in-fluences the different filament injection rates. Using too little speed when printing with these polymers may cause the material in the filament to obstruct and jam in the extruded outlet.
[0037] According to aspect 6 the controller unit and power supply located at down of structural box and all electrical input and output would connect to this part.
[0038] According to aspect 7 the incubator chamber control system that control temperature, humidity and amount of input Co2 is located in this part.
[0039] According to aspect 8 can be defined such that rotating disk unit.
All attachments and nozzles are attached on disk to create and inject fluids layer by layer scaffolds. Op-tionally this rotating disk could be substitute with another rotating disk that contains actuators such as laser and atmospheric plasma tesla mechanisms. Optionally ,On the rotating Disk with screw mechanism there is ability to add or remove the nozzles .With connect linear transmutation system that contain motor that coupled to power screw in the x, y axis transfer this mechanism of transmission of disk with nozzles and on a single sheet is carried out.
[0040] According to aspect 8 the mechanism of the hydro gel nozzle is used for inject com-bination of living cells (the types of differentiated stem cells or cells) and the components of hydrogels and a variety of new substrates that are liquid.
Creating compound scaffolds is difficult to keep the layers created together. The hydrogel injection nozzles are of the coaxial type, they can be mix hydrogel components with along with the growth factors and all media.
[0041] According to aspect optionally, the second category involves the use of low-level laser spray nozzles and low-level atmospheric plasma emitter nozzles to improve the structure of the scaffolds, which will be further elaborated. Atmospheric plasma atomic nozzles break down the surface bonds, and by releasing hydrogen bonds and levels of abstinence, the cells grow and propagate the differentiated stem cells). There is an ionizing environment that turns into plasma. In this equipment, the length of the plasma arc is 1-6 cm, with adjustable magnetic field and electrical conductivity of 5-20 kV. This produces a pair of positive-charge ions and negatively charged electrons at the surface and causes hydrophobic levels of these surfaces.
[0042] According to aspect10 In the rotary transmission disk mechanism, optionally, two types of nozzles and actuators are connected to it aspect 8, 9,11,12. The first is the scaffolding additive manufacturing mechanism that printing the later of scaffolds to shape the organs. The second category is a variety of mechanisms for modifying the surface structure. The first subdivision consists of four injection mechanisms, which are referred to below.
[0043] According to aspect 11, the adjustable wavelet for Infrared lamp and ultra violet is located in this area act as light actuator for crosslinking and polymerization of composites and hydro gels.
[0044] According to aspect 12 Optionally, Low-power infrared laser (wavelength 800-950 nm, 200 MW power, radiation dose up to 4 Jules per centimeter, and with the control of the irradiation time and according to the laser tube mounted on the devices can be changed and upgraded. Provided that the power supply of the device is also adapted to the laser tube, this laser is a potentiometer for adjusting power and output wattage.
Also, the driver can be equipped with a variety of diode types with different powers, as well as a TTL control port to control the laser diode's power. It has adjustment of values of power that could stimulate and increase proliferation and integration cells.
Low-power laser treatment is effective in the formation of organs.
[0045] According to aspect 13 the small fan located at top of extruder that decreases the temperature of heater of extruder to control filament temperature.
[0046] According to aspect 14 the connecting rod used for adjust the movement in x axes and control the vibration in the movement is this axes.
[0047] According to aspect 15, the next mechanism is the electro spinning mechanism for creating scaffolds containing nano fibers for isolation and differentiation of tissues.
This system is a fiber production method which uses electric force to draw charged threads of polymer solutions or polymer melts up to fiber diameters when high voltage is applied to a liquid droplet, the body of the liquid becomes charged, and electrostatic repulsion counteracts the surface tension and the droplet is stretched with a connection to the earth (ground).
[0048] According to aspect 16 the movement in Z axis is used by the heat plate bed in which the sample of the tissue is printed bed. The direction of the vertical axis is the motion of the crankshaft, which is raised by the linear bearings and the conductor rod, it decrease vibration of the system and the precision of the transfer is obtained through this structure.
[0049] According to aspect 16 this mechanism on injection of polymers in electro spinning is used for the production of nano fibers with core-shell structures. This nozzle is two-sided or coaxial nozzles to inter polymer and drugs made by stainless steel can be auto cleavable. The polymer solution or melt sprayed from a jet with a diameter in the range of millimeters at the high voltage applied supersedes load, then this charged fluid is affected by the passage of the electric field and the accumulation of load on the jet surface causes a jet A fluid with a diameter of millimeters or nm will be converted to create a scaffold. The printed scaffold will be connected to the inheritance, and due to the insolubility of the polymers, the possibility of coating on the polymer and composite scaffolding is possible.
[0050] According to aspect 17, the power inputs and the system wires are inserted from the bottom of the printer and connected to the controller of the device.
[0051] According to aspect 18, the output of variable volumetric syringes is pumped with adjustable output diameters. The output of fluid from injection pump is inserted into the bottom of the printer and then connected to the nozzles with disposable tubes.
[0052] According to aspect 19 all nozzles and actuators have the ability to assemble and dis-assemble, and therefore the number of these devices can be increased and decreased based on the specific printing process.
[0053] According to aspect 20, 22 the entire transfer system and nozzles are arranged in an isolated chamber like incubator condition with controlling of amount of temperature e, humanity, carbon dioxide and oxygen. Air intakes and the amount of carbon dioxide is measured and the blower is done on them on chamber via input tube. The interior control of temperature is done with two heater elements that increased and with fan it is decrease. Amount of humidity and oxygen also controlled and it is an essential condition for the survival of cells in the interior of the device during printing. The structure of printer chamber is made by double-glazed compartment.
100541 According to aspect 21, the entire printing program is controlled by computer program, the mobile app and manually. In the software menu panel, G Codes would be generated for layer by layer formation process.
[0055] According to aspect 22 ,these lead and screw mechanism act as linear actuator is used to attach the syringe by attaching the axial compressive force to the arm.
100561 According to aspect 23 , 24 This injection pumps consist of a lead and screw with size 20 millimeter and 50 centimeter long with pitch .08mi1ime1er act as power transmitting for linear movement mechanism with coupling to a stepper motor with a 1.8 degree step angel and 1.65 amperes.
[0057] According to aspect 25, 26 various types of syringes of different dimensions (0.1-30 CC) can be used. By adding the cap and adjusting the screw to the syringe diameter, these syringes are fixed within the pump mechanism.
[0058] According to aspect 27, the entire injection pump mechanism is portable and can be transported with a carrier handle along with a bio-printer.
[0059] According to aspect 28 the linear bearing mechanism and guide rod for moving and preventing vibrations are embedded in this system.
[0060] According to aspect 29 filament bases is located in top of printer that all rolled filament could inserted to extruder form this area.

[0061] According to 30 the 1.8 stepping angel stepper motor is connecting to extruder mechanism to deliver filament to melting chamber .Inside this chamber is sterilize at first of operation with Ultra violet lamp.
[0079] Reference will now be mail in detail to various exemplary embodiments of the invention. It is to be understood that the following of exemplary embodiment is not reader more detailed understanding of certain aspect and features on the invention.
[0080] In one embodiment, the present invention provide a incubator chamber contain 3d hybrid bio printer. The incubator chamber is sized such that it is adapted to surround or encompass the hybrid bioprinter while minimizing additional space. The inventive In-cubation chamber provide zone of different adjustable percentage amount of 02, N, Co2. Humid air and air surrounding the hybrid bio printer .In another embodiment, the controllers of hybrid bio printer and gas and humid air located at under plate the below the chamber isolated plates .In another embodiment, the gas and humid air inter chamber with plastic nozzle that located in bottom of chamber that connected to plastic flexible pipe .The temperature of incubator chamber is set to be between 36-38 degree centigrade and the thermal sensor detect temperature and the exact temperature could reach with increase it with heater and decrease it two fans and could control the amount of temperature in chamber. The amount of Co2, 02 ,N is measured by sensor and with insert of these gases throw inlet pipe to nozzle the set amount would reaches and it would increase cell vitality.
[0081] Thus, in some embodiment the hybrid bio printer's incubator chamber is smaller than 2 m^3 with integrated all gas and humid hair supply units.
[0082] In certain embodiments, the incubator chamber is on standard laboratory bench witch include hybrid bio printer components inside.
[0083] In alternative embodiments, this system could accommodate bioprinter isolation chamber of variety of other configuration, non-limiting examples of which are described in U.S. Patent Ns 20110136162, WO 201740675 and WO 2017/040675A1.
[0084] In Embodiments, the hybrid bio printer contain all components such as , stepper motors, Power movement displacement mechanism, All substrates for printing, nozzles , actuators, laser, plasma, controller, incubator chamber, Door with anti UV
coat, Fan ,electrical boards. cables , pipes.
[0085] In various embodiments, Material plasma treated for hydrophobicity is known art, for example U.S 5028453.
[0086] In embodiment, Micro channel and tunnel is used in micro transfer of media and this system in manufactured by electro spinning and lithography simultaneously.
[0087] In alternative embodiments example is described in US 7195733 used flow mimicking reservoir for replacing micro channels seeded with cells .The example of electro spinning in bioprinter for scaffold generation is described in U520090208577.

[0088] In alternative embodiments, this system could accommodate bioprinter of variety of other configuration , non-limiting examples of which are described in U.S.
Patent Ns.
8241905, 9149952, 20130302872. This invention contemplates that these nozzles and actuators can be configured to any bio printer known in art, as well as future im-provements in 3D bioprinter technologies.
[0089] An exemplary bioprinting system is described in PCT/US 2004/015316, US
20110136162, CN 104921840B, WO 2014194180,W02017184839A1 all incorporated herein in its entirety.
[0090] In Embodiments, rotating disk is added to bioprinter movement mechanism to change tools such as different size of nozzles, actuators, Ultra Violate and Infra-Red lamps and LED. These tools could be attachable and several injection mechanisms could add to this instrument. These UV and IR could use for photo crosslinking of hydro gels.
[0091] Different nozzles like coaxial nozzles are used to inject hydrogels and other liquid contents.
[0092] In various embodiment, tissue utilizing UV cross linking are known art, for example PCT/US2013/036479.
[0093] In alternative embodiments, this system could accommodate moveable tool changing disk of variety of other configuration, non-limiting examples of which are described in KR 101817429 Bl.
[0094] The present invention has been described with reference to particular embodiments having various features. In light of the disclosure provided above, it will be apparent to those skilled in the art that various modifications and variations can be made in the practice of present invention without departing from scope or spirit of invention. One skilled in the art will recognize that the disclosed features may be used singularly, in any combination, or omitted based on requirements and specifications of a given ap-plication or design. When an embodiment refers to "comprising" certain features, it is to be understood that the embodiments can alternatively 'consist of" or "consist es-sentially of" any one or more of the features. Other embodiments of invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention.
[0095] It is noted in particular that where a range of values is provided in this specification, each value between the upper and lower limits of that range is also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range as well. The singular form -a", "an", and "the"
include plural referents unless the context clearly dictates otherwise. It is intended that the specification and examples be considers as exemplary in nature and that variations that do not depart from the essence of the invention fall within the scope of the

12 invention. Further, all of the references cited in this disclosure are each individually in-corporated by reference herein their entries and as such are intended to provide an efficient way of supplementing the enabling disclosure of this invention as well as provide background detailing level of ordinary skill the art.
Examples [0096] This innovation could use for printing 3 layer skin. The first layer is used by bio polymer and second one is hydrogel with cells and final layer is electro spinning layer that antibiotic drug purified on it.
Brief Description of Drawings [0062] The accompanying drawings illustrate certain aspects of embodiments of present invention, and should not use to limit the invention. Together with the written de-scription the drawing serves to explain certain principles of the invention.
[0063] Fig.1 [0064] [fig.1]
is a schematic diagram showing a front view of the bio printer technology according to an embodiment of the invention.
[0065] Fig.2 [0066] [fig.2]
is a schematic diagram showing a design of side view of the entire bio-printer system.
[0067] Fig.3 [0068] [fig.31 is a schematic diagram presentation a design of top view of the entire bio-printer system.
[0069] Fig.4 [0070] [fig.4]
is a schematic diagram illustrates a detailed configuration of the bio printer according to an embodiment of the invention.
[0071] Fig.5 [0072] [fig.51 is a schematic diagram showing a top view of the injection pump mechanism according to an embodiment of the invention.
[0073] Fig.6 100741 [fig.61 is a schematic diagram showing a side view of the injection pump mechanism according to an embodiment of the invention.
1100751 Fig.7

13 [0076] [fig.7]
is a schematic diagram presenting a front view of the injection pump mechanism according to an embodiment of the invention.
[0077] Fig.8 [0078] [fig.8]
is a schematic diagram illustrates detailed configuration of the injection pumps according to an embodiment of the invention.
Industrial Applicability [0097] This invention could use to clone hard and soft organ with surface treatment and could implements in regenerative medicine industry.
Citation List [0098] Citation List follows:
Patent Literature [0099] PTL 1: U.S. Patent 20110136162 [0100] PTL 2: PCT/US2013/036479 [0101] PTL 3: WO 2017/040675A1 [0102] PTL 4: KR 101817429 B 1 101031 PTL 5: U.S. Patent Ns. 9149952 [0104] PTL 6: U.S. Patent Ns. 8241905 [0105] PTL 7: U.S. Patent Ns. 20130302872

Claims

AMENDED CLAIMS
received by the International Bureau on 12 December 2019 (12 12 2019) [Claim 11 Designing hard and soft human organs for the purpose of copying and printing all organs according to automatic computerized segmentation based on the anatomy, density and physiology of the tissues. Designing of physical components of an automatic multi-stage three-dimensional system.
[Claim 21 Manufacturing of automatic multi-stage three-dimensional system to clone organs by injection of engineering scaffolds of individual tissue or in combination with the use of All types of bio-polymers, nano-composites, all kinds of hydrogels containing stem cells, cells and Media and drugs.
[Claim 31 The Designing and construction of a claim 1 , 2 system, the components of which are:
a double layer rectangular structural body a linear motion mechanism for transferring components in the axes x, y, z a heat bed plate, an Incubator chamber, a rotating disk with a attachable plate for nozzles and actuators , a coaxial injection nozzle that ejecting hydro gels components with cells and Media , an Electro spinning system , a Coaxial injection nozzle, a bio composite and polymer injection nozzles, a Rotating disk, an Extruder of filaments, a power controlled Low-power laser and plasma disposition , Housing and whirling, [Claim 41 The construction of machine in the claim 2 has the ability to modify the structure of scaffolding created by a low-power laser nozzle with a variable amplitude connected to the displacement disk of the nozzles claim 2 with variable power control circuit feature.
[Claim 51 The construction of the claim 2 has the ability to modify the structure of the scaffolding created by the nozzle of the atmospheric plasma mechanism attached to the nozzle plate shown.
[Claim 61 Manufacturing of claim 2 that can control the conditions of tissues in chamber with incubator conditions in the internal printing space, these conditions are including controlled amount of temperature, humidity, AMENDED SHEET (ARTICLE 19) oxygen and carbon dioxide in certain interval.
[Claim 71 The construction of a claim 2 machine, containing four ranged control ultra violet lamps, is used at the top of the injection chamber for ster-ilization and polymerization purposes.
[Claim 81 Manufacturing of External smart Injection pumps that pumps different composite, hydrogels, ceramics and other liquids containing cells or growth factors with different viscosities with adjustable pressure and injection feed rate. This pump can inject different from different syringes shape and size with controlling of temperature of syringes.
This pumps comprising mechanism that consists of these parts:
Housing with normal or luer lock taper, wiring, Stepper motors, Coupling, Structural body, Lead screw mechanism, Heat and cold elements, Thermal Sensor, AMENDED SHEET (ARTICLE 19)