Device for skin bioprinting and method of manufacturing device for skin bioprinting

Abstract

The present invention provides an apparatus for skin bioprinting and a method of manufacturing an apparatus for skin bioprinting, the apparatus comprising: a dispenser module configured to drive and dispense bio-ink from a first chamber and to drive and dispense a cross-linking agent from a second chamber; a storage module connected to the dispenser module, the storage module including a first chamber storing bio-ink and a second chamber storing a cross-linking agent; a mixing module connected to the storage module, the mixing module mixing the bio-ink and the crosslinking agent dispensed from the first and second chambers, respectively; and a rotatable applicator connected to the mixing module, the rotatable applicator rotatably applying a mixture of bio-ink and cross-linking agent from the mixing module to a surface of a patient in need of skin regeneration.

Description

Technical Field

[0001] The present disclosure relates broadly to devices for skin bioprinting and methods of making devices for skin bioprinting. Background Art

[0002] Treatment of patients with skin diseases and conditions such as burns may involve transplantation of cultured epithelial autograft sheets, or more commonly, skin grafts. The former requires relatively large numbers of cells, long culture times, and the resulting sheets tend to be brittle. The latter results in donor site morbidity and may not be aesthetically pleasing due to the need to "mesh" the skin by flattening and cutting slits of the skin to achieve coverage of a larger area.

[0003] Bioprinters have proven to be a useful tool for printing tissue constructs and could potentially bridge the gap between organ shortages and transplant needs. Typically, bioprinting involves recreating the three-dimensional (3D) structure of tissues to mimic the natural architecture of the extracellular matrix in which cells are suspended. In addition, cells can also be introduced into these constructs.

[0004] However, current state-of-the-art bioprinters tend to be complex and expensive devices. For example, some bioprinters may have printing components (e.g., print heads) of considerable size and therefore may not be suitable for use as handheld devices due to their weight and limited maneuverability. Other forms of print heads may have straight-edged print heads or print heads with flat rectangular surfaces, which may only work well on relatively flat surfaces but may have difficulty dispensing material on contoured body parts such as the nose, ears, and armpits.

[0005] Therefore, there is a need for devices for skin bioprinting and methods of making devices for skin bioprinting that seek to solve or at least ameliorate one of the above-mentioned problems. Summary of the invention

[0006] In one aspect, a device for skin bioprinting is provided, comprising: a dispenser module configured to drive and dispense bio-ink from a first chamber, and to drive and dispense a cross-linking agent from a second chamber; a storage module coupled to the dispenser module, the storage module comprising a first chamber for storing the bio-ink and a second chamber for storing the cross-linking agent; a mixing module coupled to the storage module, for mixing the bio-ink and the cross-linking agent dispensed from the first and second chambers, respectively; and a rotatable applicator coupled to the mixing module, for rotatably applying the mixture of the bio-ink and the cross-linking agent from the mixing module to the surface of a patient in need of skin regeneration.

[0007] In one embodiment, the apparatus further comprises a cooling module configured to contain a cooling element for cooling the bio-ink, the cooling being to prevent premature gelation of the bio-ink.

[0008] In one embodiment, the cooling module is arranged adjacent to or at least partially surrounding the mixing module such that, in use, the cooling element contained therein is able to cool the mixture of the bio-ink and the cross-linking agent.

[0009] In one embodiment, the dispenser module includes a first piston configured to be displaced within a first chamber to dispense bio-ink from the first chamber and a second piston configured to be displaced within a second chamber and dispense a crosslinker from the second chamber.

[0010] In one embodiment, the dispenser module further comprises one or more pneumatic inlets configured to introduce gas for driving the first and / or second piston.

[0011] In one embodiment, the dispenser module further includes a plunger coupled to the first and second pistons.

[0012] In one embodiment, the mixing module comprises a static mixer.

[0013] In one embodiment, the rotatable applicator includes a roller ball configured to roll along the surface of a patient in need of skin regeneration.

[0014] In one embodiment, the rotatable applicator includes a cylindrical roller configured to roll along the surface of a patient in need of skin rejuvenation.

[0015] In one embodiment, the apparatus further comprises a control unit configured to control the dispensing of the bio-ink and the cross-linking agent from the first and second chambers, respectively, to the mixing module.

[0016] In one embodiment, the control unit is further configured to control mixing of the bio-ink and the cross-linking agent in the mixing module.

[0017] In one embodiment, the device further comprises an activation switch electrically coupled to the control unit for activating and / or deactivating the control unit.

[0018] In one embodiment, the device further comprises a pneumatic pump coupled to the one or more pneumatic inlets.

[0019] In one embodiment, the device is a handheld device.

[0020] In one embodiment, the storage module is removably coupled to the dispenser assembly.

[0021] In one embodiment, the mixing module is removably coupled to the storage module.

[0022] In one embodiment, the first chamber and the second chamber of the storage module are independently removable.

[0023] In one embodiment, the cooling module comprises a cooling element.

[0024] In one embodiment, the cooling element comprises a phase change material.

[0025] In one aspect, a method for manufacturing an apparatus for skin bioprinting is provided, the method comprising: providing a dispenser module configured to drive and dispense bio-ink from a first chamber, and to drive and dispense a cross-linking agent from a second chamber; providing a storage module coupled to the dispenser module, the storage module comprising a first chamber for storing the bio-ink and a second chamber for storing the cross-linking agent; providing a mixing module coupled to the storage module for mixing the bio-ink and the cross-linking agent dispensed from the first and second chambers, respectively; and providing a rotatable applicator coupled to the mixing module for rotatably applying the mixture of the bio-ink and the cross-linking agent from the mixing module to a surface of a patient in need of skin regeneration.

[0026] definition

[0027] As used herein, the term "biocompatible" should be broadly interpreted to refer to the ability of a material to perform its intended function without inducing a significant inflammatory response, immunogenicity, or cytotoxicity in native cells, tissues, or organs.

[0028] As used herein, the term "biologically inert" should be broadly interpreted to refer to materials that do not substantially elicit an immune response in humans or animals when placed in or in contact with a biological environment in vivo.

[0029] The term "bio-ink" as used herein should be broadly interpreted to refer to a composition comprising a hydrogel for bioprinting or a composition comprising a mixture of cells and a hydrogel for bioprinting.

[0030] As used herein, the term "crosslinker" should be broadly interpreted as referring to an agent (e.g., compound, composition) that can cause gelation of a bio-ink. For example, the term "crosslinker" can include an agent that can chemically link macromolecules using covalent or ionic bonds. For example, the term "crosslinker" can include an agent that can change the pH of a bio-ink. Therefore, as used herein, the terms "crosslink" and "crosslinking" refer to the step of causing gelation of a bio-ink.

[0031] The term "hydrogel" as used herein should be broadly interpreted to refer to a network of hydrophilic polymers cross-linked by covalent or non-covalent bonds. Due to the hydrophilicity of the hydrogel components, the hydrogel swells by absorbing water from an aqueous solution, but does not dissolve because of its cross-linked structure.

[0032] As used herein, the term "dynamic mixer" should be broadly interpreted to refer to an in-line mechanical device having at least one movable mixing element, designed to provide continuous mixing of fluids (eg, gases or liquids).

[0033] The term "static mixer" as used herein should be broadly interpreted as referring to an online device having at least one fixed / static / immovable (e.g., in a translational manner) mixing element, designed to provide continuous mixing of fluid (e.g., gas or liquid). Static mixers may include a series of immovable (e.g., in a translational manner) mixing elements arranged in a housing. Mixing elements may be spiral elements and / or baffles, etc. The housing and mixing elements of a static mixer may be independently made of metal or plastic. Typical materials of static mixer components include, but are not limited to, stainless steel, polypropylene, polyaldehyde, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF) and polyvinyl chloride (PVC).

[0034] As used herein, the term "substrate" should be broadly interpreted to refer to any supporting structure.

[0035] The term "layer", when used to describe a first material, should be broadly interpreted as referring to a first depth of the first material, which can be distinguished from a second depth of the second material. The first material of the layer can exist as a continuous film, as a discontinuous structure, or a mixture of the two. The layer can also have a substantially uniform depth throughout or at different depths. Thus, when the layer is formed of individual structures, the size of each individual structure can be different. The first material and the second material can be the same or different, and the first depth and the second depth can be the same or different.

[0036] The term "continuous" when used to describe a film or layer should be broadly interpreted to refer to a film or layer that has substantially no gaps, holes or voids throughout the film or layer. In this regard, a continuous film or layer is also intended to include a film or layer that may have minor gaps or holes or voids that may not significantly affect the desired properties of the film or layer.

[0037] As used herein, the term "micro" should be broadly interpreted to include sizes from about 1 micron to about 1000 microns.

[0038] As used herein, the term "nano" should be broadly interpreted to include dimensions less than about 1000 nm.

[0039] The term "particle" as used herein refers broadly to a discrete entity or discrete body. The particles described herein may include organic, inorganic or biological particles. The particles described herein may also be large particles formed by aggregates of multiple sub-particles or fragments of small objects. The particles of the present disclosure may be spherical, substantially spherical or non-spherical, such as irregularly shaped particles or ellipsoidal particles. The term "size" when used to refer to a particle broadly refers to the maximum dimension of the particle. For example, when the particle is substantially spherical, the term "size" may refer to the diameter of the particle; or when the particle is substantially non-spherical, the term "size" may refer to the maximum length of the particle.

[0040] Unless otherwise stated, the terms "coupled" or "connected" as used in this specification are intended to encompass either a direct connection or a connection through one or more intermediate means.

[0041] As used herein, the term "associated with" when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to, a physical, chemical, or biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly connected to each other, or element A may contain element B, or vice versa.

[0042] As used herein, the term “adjacent” when referring to two elements means that one element is not far from another element, and may be, but is not limited to, the elements contacting each other, or may further include elements separated by one or more additional elements located therebetween.

[0043] The term "and / or", such as "X and / or Y", is understood to mean "X and Y" or "X or Y", and should be considered to provide clear support for both or either meanings.

[0044] In addition, in this specification, whenever used, the word "substantially" is understood to include, but is not limited to, "all" or "completely", etc. In addition, terms such as "including", "comprising", etc., whenever used, are intended to be non-restrictive descriptive language, because in addition to other components that are not explicitly cited, they broadly include the elements / components recorded after such terms. For example, when "including / comprising" is used, mentioning "one" feature is also intended to mention "at least one" of the feature. In the appropriate context, terms such as "consisting", "consist", etc. can be regarded as subsets of terms such as "including / comprising", etc. Therefore, when using terms such as "including / comprising" etc. in the embodiments disclosed herein, it will be appreciated that these embodiments provide teachings of corresponding embodiments using terms such as "comprising", etc. Additionally, terms such as "about," "approximately," etc., whenever used, typically mean a reasonable variation, for example, a variation of + / - 5% of the disclosed value, or a variation of 4% of the disclosed value, or a variation of 3% of the disclosed value, a variation of 2% of the disclosed value, or a variation of 1% of the disclosed value.

[0045] In addition, in this specification, certain values ​​may be disclosed in a range. The values ​​showing the endpoints of the range are intended to illustrate the preferred range. Whenever a range is described, the range is intended to cover and teach all possible sub-ranges and individual numerical values ​​within the range. That is, the endpoints of the range should not be interpreted as rigid limitations. For example, the description of the 1% to 5% range is intended to specifically disclose sub-ranges 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., as well as to disclose values ​​within the range separately, such as 1%, 2%, 3%, 4% and 5%. It should also be understood that individual numerical values ​​within the range also include integers, fractions and decimals. In addition, whenever a range is described, the range is also intended to cover and teach values ​​or significant figures (where appropriate) up to 2 additional decimal places starting from the displayed numerical endpoints. For example, description of a range of 1% to 5% is intended to specifically disclose the range 1.00% to 5.00% and 1.0% to 5.0% and all intermediate values ​​thereof across that range (e.g., 1.01%, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1%, 1.2% ... 4.8%, 4.9%, 5.0%, etc.). The above specific disclosure meaning applies to any depth / breadth of the range.

[0046] In addition, when describing some embodiments, the present disclosure may have disclosed methods and / or processes as a specific sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the disclosed specific sequence of steps. Other step sequences are possible. The specific order of steps disclosed herein should not be interpreted as overly restrictive. Unless otherwise required, the method and / or process disclosed herein should not be limited to the steps performed in the written order. The order of the steps can vary, but still remain within the scope of the present disclosure.

[0047] Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more features / characteristics discussed herein, one or more of these features / characteristics may also be excluded in other alternative embodiments, and the present disclosure provides support for such exclusions and these related alternative embodiments. DETAILED DESCRIPTION

[0048] Disclosed below are exemplary, non-limiting embodiments of devices for skin bioprinting, methods of making devices for skin bioprinting, and methods of applying a mixture of bio-ink and cross-linking agent.

[0049] Device for skin bioprinting

[0050] In various embodiments, an apparatus for skin bioprinting is provided, comprising: a dispenser module configured to drive and dispense bio-ink from a first chamber, and to drive and dispense a cross-linking agent from a second chamber; a storage module coupled to the dispenser module, the storage module comprising a first chamber for storing the bio-ink and a second chamber for storing the cross-linking agent; a mixing module coupled to the storage module, for mixing the bio-ink and the cross-linking agent dispensed from the first and second chambers, respectively; and a rotatable applicator coupled to the mixing module, for rotatably applying the mixture of the bio-ink and the cross-linking agent from the mixing module to a surface of a patient in need of skin regeneration.

[0051] In various embodiments, the device can be a handheld bioprinter designed to deposit a mixture of a bioink and a crosslinker (e.g., a cell-laden bioink) directly onto a surface (e.g., a wound bed). By coupling a mixing mechanism (mixing a bioink base with (one or more) crosslinkers) to a rotatable applicator (e.g., a roller ball assembly), the device can advantageously achieve uniform deposition of the mixture of bioink and crosslinker onto a contoured surface, such as a human body.

[0052] In various embodiments, the components of the device (e.g., a dispenser module, a storage module, a mixing module, and a rotatable applicator) can all be built into the device and cannot be disassembled. In various embodiments, one or more components of the device (e.g., a dispenser module, a storage module, a mixing module, and / or a rotatable applicator) can be detachable from the device. In various embodiments, one or more components of the device (e.g., a dispenser module, a storage module, a mixing module, and / or a rotatable applicator) can be disposable and replaceable. The modularity of the device can allow individual components to be replaced independently of each other. This can advantageously minimize cross-contamination when the device is reused, because components in contact with biological matter (e.g., a patient's cells) can be disposed of and replaced after use. In various embodiments, the simple construction of the device can contribute to the easy replacement of its various components compared to more complex bioprinting devices known in the art (whose components are obviously not disposable due to their complex construction).

[0053] In various embodiments, one or more components of the device are biocompatible, bioinert and sterilizable. In various embodiments, the parts of the device contacted with the patient's surface (directly or indirectly) are biocompatible, bioinert and sterilizable. In various embodiments, the whole device is biocompatible, bioinert and sterilizable. Suitable materials and techniques known in the art can be used for sterilization, such as ethylene oxide, gamma rays or electron beam irradiation, plasma or autoclave. Therefore, one or more components of the device are made of materials that can withstand the desired sterilization method without causing substantial or obvious losses to their desired properties or functions. In various embodiments, the device can be substantially free of any electronic components. This can advantageously promote simplified sterilization, because the device can be modified to select sterilization techniques more.

[0054] In various embodiments, the patient in need of skin regeneration or skin transplantation can be a patient with a skin disease or condition. In various embodiments, the patient in need of skin regeneration can have damaged or missing skin that cannot heal on its own. In various embodiments, the skin disease or condition can be a burn, infection, cancer, for example, skin loss caused by surgical removal of skin cancer, skin ulcers, pressure injuries, such as bedsores, slow-healing wounds or very large wounds, such as open fractures that destroy the skin. In various embodiments, the burn can be a friction burn, a cold burn, a heat burn, a radiation burn, a chemical burn, an electrical burn or a mixture thereof. In various embodiments, the burn can be classified based on the degree of skin injury, i.e., a 1st degree burn, a 2nd degree burn, a 3rd degree burn or a 4th degree burn. Advantageously, the device for skin bioprinting can deposit a substantially continuous layer of bio-ink (e.g., a bio-ink loaded with keratinocytes) directly onto the surface (e.g., dermis) of the patient, thereby achieving better cosmetic results for the patient. In various embodiments, the device for skin bioprinting is optionally used to apply the mixture to a tissue culture surface / substrate for in vitro tissue culture. Thus, in various embodiments, the device can be used in applications such as skin bioprinting, wound healing, burns, chronic wound management, fabrication of in vitro tissue models, deposition of non-collagen bio-inks (such as alginate and calcium salts), etc.

[0055] case

[0056] In various embodiments, the device further includes a housing / cover. The housing can include an outer surface for allowing an operator, such as a healthcare professional to hold / grip the device, and a chamber defined in the housing for accommodating various components of the device, such as a dispenser module, a storage module, a mixing module, and a rotatable applicator. In various embodiments, the housing can be a single integrated housing. In various embodiments, the housing can be a plurality of separate housings for each module, and the plurality of separate housings can be assembled together to form the device. In various embodiments, the housing can further include one or more outlets / openings for allowing various components of the device, such as a rotatable applicator, to enter or be installed inside the housing from outside the housing. The various components of the device can be detachable and replaceable. The various components of the device can be arranged to move independently of each other and / or move in a coordinated manner in the housing.

[0057] In various embodiments, the device can be a handheld device, that is, its shape and size can be held by a human hand. The housing can have a specific shape and / or form that meets ergonomic purposes, facilitating the operator, such as a healthcare professional, to manipulate. In various embodiments, the device can include one or more controls placed on the outer surface of the housing, such as electronic controls, physical controls (e.g., buttons, knobs, sliders, switches, levers, joysticks, etc.). Each of the one or more controls can be associated with a component of the drive device, such as a dispenser module. The drive of the component can include the movement, position and / or orientation of the control component. The control of the drive can be physical, electronic, manual and / or automatic. The housing of the device can be configured ergonomically to enhance the user experience (e.g., easy to finely control, manipulate and handle) and improve the comfort during the use of the device. The housing of the device can be symmetrical, so that both right-handed and left-handed users can use it. One or more controls can be placed on the outer surface of the housing in a manner that is easy to touch and / or easy to push / press smoothly, thereby almost no need or no need for the operator to reposition the grip / grip position of the device.

[0058] Control unit

[0059] In various embodiments, the device may further include a control unit. In various embodiments, the control unit may be configured to control the distribution of the bio-ink and the cross-linking agent from the first and second chambers to the mixing module, respectively. In various embodiments, the control unit may be further configured to control the mixing of the bio-ink and the cross-linking agent in the mixing module. In various embodiments, the device may further include an activation switch electrically coupled to the control unit for activating and / or deactivating the control unit. In various embodiments, the control unit and / or the activation switch may be placed in a handheld portion of the device (e.g., as a finger trigger) or in a pneumatic pump (e.g., as a foot switch).

[0060] Storage Module

[0061] In various embodiments, the storage module functions to contain / accommodate the bio-ink and cross-linking agent prior to mixing and dispensing from the device. In various embodiments, the first and second chambers can each take the form of an elongated container having a first end and a second end opposite the first end. The elongated container can have a suitable cross-sectional shape, such as circular, semi-circular, elliptical, semi-elliptical, square, or rectangular. The first and second chambers can each include at least one opening at a first end thereof, and at least one opening at a second end thereof.

[0062] In various embodiments, the openings at the respective first ends of the first and second chambers can be configured to be coupled to a dispenser module to facilitate the transfer of a driving force from the dispenser module to the bio-ink in the first chamber and the cross-linking agent in the second chamber. In various embodiments, the openings at the respective second ends of the first and second chambers can be configured to be coupled to a mixing module to facilitate the dispensing of the bio-ink from the first chamber and the cross-linking agent from the second chamber.

[0063] In various embodiments, the storage module can be removably coupled to the dispenser module / assembly. In various embodiments, the storage module can be removably coupled to the mixing module. In various embodiments, the first chamber and the second chamber of the storage module can be independently movable. In various embodiments, the openings at the first end and the second end of the chamber can include connectors that facilitate removable coupling of the first and second chambers to the dispenser module and the mixing module, respectively. In various embodiments, the first chamber and the second chamber of the storage module can be disposable and replaceable. In various embodiments, the first chamber and the second chamber of the storage module can be in the form of a disposable box. Advantageously, once the existing box of bio-ink and cross-linking agent is exhausted, a new box of bio-ink and cross-linking agent can be inserted into the device.

[0064] Hybrid Module

[0065] In various embodiments, the function of the mixing module is to mix the bio-ink and the cross-linking agent dispensed from the first and second chambers, respectively. In various embodiments, the mixing of the bio-ink and the cross-linking agent causes the bio-ink to undergo gelation. Advantageously, pre-mixing with the mixing module can ensure that the materials (e.g., bio-ink and cross-linking agent) are uniformly mixed before application / dispensing. In addition, pre-mixing with the mixing module can promote better healing and regeneration of the skin surface. In various embodiments, the mixing module can be disposable and replaceable. Advantageously, by having a disposable and replaceable mixing module, cross contamination can be minimized when reusing the device.

[0066] In various embodiments, the mixing module may include a mixing chamber, a mixer disposed within the mixing chamber, at least one opening at a first end, and at least one opening at a second end opposite the first end of the mixing module.

[0067] In various embodiments, the mixing module can include two openings at a first end. In various embodiments, each opening at the first end of the mixing module can be configured to be coupled to an opening at a second end of the first and second chambers of the storage module to receive the bio-ink and the cross-linking agent, respectively. In various embodiments, the mixing module can include an opening (e.g., a nozzle) at the second end that is configured to distribute the mixture of the bio-ink and the cross-linking agent to the rotatable applicator.

[0068] In various embodiments, the mixer can be a combination of a static mixer and a dynamic mixer. In various embodiments, the mixer can be just a dynamic mixer. In various embodiments, the mixer can be just a static mixer. The inventors have recognized that, in various embodiments, a mixing module consisting only of a static mixer can be sufficient to quickly achieve uniform mixing of the bio-ink and the crosslinking agent. Using a static mixer alone can also advantageously provide a simpler device construction by reducing unnecessary volume (e.g., additional mechanical and electronic components used to configure the dynamic mixer) and allow the device to be handheld.

[0069] Rotatable applicator

[0070] In various embodiments, the function of the rotatable applicator is to apply the mixture of the bio-ink and the cross-linking agent from the mixing module to the surface of the patient in need of skin regeneration. In various embodiments, the mixture of the bio-ink and the cross-linking agent is in the form of a gel when applied to the surface. In various embodiments, the rotatable applicator can be capable of applying a substantially uniform layer of the bio-ink and cross-linking agent mixture. In various embodiments, when applying the mixture of the bio-ink and the cross-linking agent, the rotatable applicator can be directly or indirectly in contact with the skin surface of the patient. In one example, the rotatable applicator can contact and apply the mixture of the bio-ink and the cross-linking agent directly to the skin surface of the patient (i.e., direct application). In another example, the rotatable applicator can contact and apply the mixture of the bio-ink and the cross-linking agent to a dressing (e.g., a skin regeneration template) placed on the surface of the patient (i.e., indirect application).

[0071] In various embodiments, the rotatable applicator may include a ball roller configured to roll along the surface of a patient in need of skin regeneration. In various embodiments, the ball roller may be configured to rotate around two or more axes of rotation (e.g., 3 axes). In various embodiments, the ball roller may promote omnidirectional deposition of a mixture of materials, such as bio-ink and a cross-linking agent. In various embodiments, the rotatable applicator may additionally include a ball roller adapter for coupling the ball roller to the nozzle of the mixer.

[0072] In various embodiments, the ball bearings can advantageously allow a relatively large surface area to be covered in a relatively short time compared to nozzles used to dispense a mixture of a material, such as a bio-ink and a cross-linking agent. In various embodiments, the ball bearings can advantageously allow material to be more easily deposited on contoured surfaces (e.g., contoured body surfaces such as the nose and ears). In various embodiments, the use of ball bearings in a bioprinter can be particularly relevant in the context of a handheld device that can be easily manipulated over a contoured surface (e.g., of human anatomy) and that can provide the physical contact necessary for a rolling action to evenly distribute the mixture of the bio-ink and the cross-linking agent. On the other hand, the inventors have recognized that for a print head mounted on an XYZ Cartesian stage (like most bioprinters), physical contact with a contoured surface cannot be easily maintained, making the use of the print head irrelevant and impractical.

[0073] In various embodiments, the rotatable applicator may include a cylindrical roller configured to roll along the surface of a patient in need of skin regeneration. In various embodiments, the cylindrical roller may be configured to rotate around a single axis of rotation. In various embodiments, the rotatable applicator may additionally include a cylindrical roller adapter for coupling the cylindrical roller to the nozzle of the mixer. In various embodiments, the cylindrical roller adapter may include an axle that allows the cylindrical roller to be mounted thereon.

[0074] In various embodiments, the ball adapter and cylindrical roller adapter can include one or more hollow compartments to allow for the inclusion of a cooling element therein that provides cooling to the mixture of the bio-ink and the cross-linking agent. Thus, in various embodiments, the cooling module can be placed within the adapter of the rotatable applicator. Since the cooling module can at least partially surround the mixing module, in various embodiments, at least a portion of the mixing module can also be placed within the adapter of the rotatable applicator.

[0075] In various embodiments, the ball and cylindrical roller can be capable of applying the mixture of bio-ink and cross-linking agent uniformly on the wavy contour of the patient's surface. In various embodiments, the ball and cylindrical roller can be configured to different sizes to adapt to the working position and surface. In various embodiments, the ball can have a diameter falling within the range of about 7.8mm to about 30mm. In various embodiments, the diameter of the ball can fall within the range of starting and ending points selected from the following groups of numbers: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30mm. Therefore, in various embodiments, the rotatable applicator can be configured to be detachable from the device, facilitating the interchange of balls and cylindrical rollers of different sizes so that balls or cylindrical rollers of sizes suitable for the working position and surface are connected to the device. For example, over the nose and ear areas, the ball or cylindrical roller may have a smaller diameter to facilitate application in such areas.

[0076] In various embodiments, the use of a ball or cylindrical roller can facilitate easier access to highly contoured surfaces on human anatomy, such as the nose, ears, and armpits. Therefore, ball and cylindrical rollers can be superior to other forms of printheads, such as straight-edge printheads or printheads with flat rectangular surfaces, which can only work well on relatively flat surfaces but may encounter difficulties dispensing material in contoured body parts such as the nose, ears, and armpits.

[0077] Distributor module

[0078] In various embodiments, the function of the dispenser module is to provide one or more driving forces for dispensing the bio-ink and the cross-linking agent from the first and second chambers, respectively. In various embodiments, the dispenser module can be a pneumatically driven dispenser module having an adjustable pressure to achieve different deposition rates. In various embodiments, the dispenser module can be configured to dispense the bio-ink and the cross-linking agent at a controllable flow rate / dispensing rate.

[0079] In various embodiments, the dispenser module may include a first piston and a second piston, the first piston being configured to displace / move within the first chamber to dispense bio-ink from the first chamber, and the second piston being configured to displace / move within the second chamber and dispense a crosslinker from the second chamber. In various embodiments, the first and second pistons may be coupled to the first and second chambers of the storage module via respective openings at the first ends of the first and second chambers. In various embodiments, the dispenser module may further include a plunger coupled to the first and second pistons, the plunger being configured to drive the first and second pistons. In various embodiments, the plunger and piston may be configured to move in tandem.

[0080] In various embodiments, the distributor module can additionally include one or more pneumatic inlets configured to introduce gas for displacing / moving the plunger and the first and second pistons. In various embodiments, the distributor module can include a single pneumatic inlet. Advantageously, the use of a single pneumatic inlet (rather than multiple control cables for motors and coolant lines in conventional bioprinters) can reduce the size of the device, making it smaller and easier to handle.

[0081] In various embodiments, one or more pneumatic inlets can be coupled to a pump. In various embodiments, the pump can be a positive displacement pump. In various embodiments, the pump can be a pneumatic pump configured to apply gas or air pressure to displace / move the plunger and piston. In various embodiments, the pneumatic dispenser module can advantageously have minimal instrumentation requirements, i.e., only compressed air is required to dispense the bio-ink and cross-linking agent.

[0082] In various embodiments, the plunger can be configured to move in response to a force applied thereto by an applied air or gas pressure, which in turn causes the first and second pistons to move in tandem. In various embodiments, the plunger and piston can be made of a material having a relatively low coefficient of friction, such as polytetrafluoroethylene (PTFE), to reduce the force required to displace the plunger and piston. This can advantageously facilitate fine control of the flow rate / dispensing rate of the bio-ink and cross-linking agent. It will be appreciated that control of the dispensing rate of the bio-ink and cross-linking agent can be achieved by other means, including the use of a motor-driven syringe. However, the inventors recognize that such other means may compromise the ease of operation of the device due to the increased size of the device. Therefore, in various embodiments, the components of the device can be selected from the perspective of realizing a handheld device that is easy for the operator to manipulate.

[0083] In various embodiments, the minimum pressure required to move the plunger and piston can fall within the range of about 0.5psi to about 20psi. In various embodiments, the minimum pressure required to move the plunger and piston can fall within the range of starting and ending points selected from the following group of numbers: 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5 and 20psi. Advantageously, a lower minimum operating pressure can allow for fine adjustment and fine control of the distribution rate / flow rate of bio-ink and cross-linking agent. It will be appreciated that the range of pressure depends on the configuration of the device. For example, the operating pressure can be increased by changing the friction of the plunger. In various embodiments, the dispenser module can be configured to dispense the bio-ink and the cross-linking agent at a flow rate falling within a range of about 5 ml / min to about 100 ml / min. In various embodiments, the flow rate of the bio-ink and the cross-linking agent can fall within a range having a start point and an end point selected from the following group of numbers: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 ml / min.

[0084] In various embodiments, the chamber of the storage module can be variable so that the bio-ink and cross-linking agent can be stored in different ratios without having to change the pneumatic settings of the pump. This ease of use can provide greater convenience when using fluids with different mixing ratios. In various embodiments, the dispenser module can be configured to provide a predictable flow rate of the bio-ink and cross-linking agent by changing the volume of the storage chamber while keeping the pressure and therefore the plunger speed the same.

[0085] In various embodiments, the dispenser module can be controlled by a control unit. In various embodiments, the dispensing of the bio-ink and the cross-linking agent from the first and second chambers can be based on user input. In various embodiments, the dispensing of the bio-ink and the cross-linking agent from the first and second chambers can be activated and / or deactivated based on user input by an activation switch electrically coupled to the control unit. In various embodiments, the control unit and / or the activation switch can be placed on the handheld portion of the device or placed in the pneumatic pump. In various embodiments, the activation switch can be a finger trigger placed on the handheld portion of the device. In various embodiments, the activation switch can be a foot switch placed in the pneumatic pump. In various embodiments, when the activation switch is activated, the bio-ink and the cross-linking agent are dispensed, mixed, and allowed to be applied / rolled onto the surface. In various embodiments, when the activation switch is deactivated, the bio-ink and the cross-linking agent are not dispensed, and they do not leak from the device.

[0086] Cooling module

[0087] In various embodiments, the device may further include a cooling module configured to include a cooling element for cooling the bio-ink, the cooling being to prevent premature gelation of the bio-ink. In various embodiments, the function of the cooling module is to provide temperature control by maintaining the bio-ink at a desired operating temperature. In various embodiments, cooling the bio-ink can prevent clogging of the mixer in the mixer module. In various embodiments, cooling the bio-ink can allow the bio-ink to be usable for a longer period of time, thereby allowing an operator, such as a healthcare professional, to have greater flexibility and wiggle room when using the device (e.g., in an operating room).

[0088] In various embodiments, the cooling module is arranged adjacent to or at least partially surrounding the mixing module so that when in use, the cooling element contained therein is capable of cooling the mixture of the bio-ink and the crosslinking agent. In various embodiments, the cooling module may include one or more compartments arranged adjacent to or at least partially surrounding the mixing module, the one or more compartments being used to contain the cooling element. In various embodiments, the cooling module includes a cooling element. In various embodiments, the cooling element may include a phase change material. Examples of phase change materials include, but are not limited to, hydroxyethyl cellulose, sodium polyacrylate, and vinyl-coated silicone. In various embodiments, the cooling element may include an electrically activated cooling element (e.g., a thermoelectric cooling element, such as a heat pump and a conductive element, etc.).

[0089] In various embodiments, the cooling module is configured to maintain the temperature of the bio-ink within a range of about 0°C to about 20°C. In various embodiments, the cooling module is typically maintained at a temperature below the temperature of the bio-ink. In various embodiments, phase change materials with different operating temperatures (e.g., -20°C, -10°C, -5°C, 0°C, 5°C, 10°C, etc.) can be used to maintain the bio-ink material at an appropriately low temperature to prevent premature gelation.

[0090] In various embodiments, the cooling module can provide a relatively simple technique for cooling bio-ink compared to complex bio-printers in the art that are driven by multiple motors, require multiple control cables, are cooled with coolant supplied through relatively large coolant lines, and have a pistol-like configuration. For existing bio-printers available in the art, bio-printers tend to have a larger base area and are heavier. They are cumbersome to operate due to thick coolant lines and cables. The pistol configuration is also clumsy. On the other hand, various embodiments of the device disclosed herein can be operated with at least one pneumatic line, an optional finger trigger line, and can be held in a pen holder, which makes it easy to manipulate. In various embodiments, cooling can be achieved by using phase change materials instead of coolant lines, which further improves its compactness and usability.

[0091] Bio-ink

[0092] In various embodiments, the bio-ink can be applied to bioprinting. In various embodiments, the bio-ink can be applied to skin regeneration. In various embodiments, the bio-ink can include natural and / or synthetic materials. In various embodiments, the bio-ink includes a polymer component. Examples of polymer components can include, but are not limited to, alginate, gelatin, collagen, such as denatured collagen, undenatured collagen, polyethylene glycol, and functionalized hyaluronic acid, and combinations thereof. In various embodiments, the polymer component can be collagen. Collagen is a major component of the extracellular matrix of tissues and can be derived from sources such as pig, cow, or fish tissue. Typically, collagen can be dissolved in acid. After mixing with an appropriate amount of alkali to neutralize the acid, the collagen forms a gel. This gelation process can be accelerated by incubation at 37°C, or slowed down by ice cooling. Other natural polymers such as alginate can also be used as bio-inks printed with handheld devices.

[0093] In various embodiments, the bio-ink may further include cells. In various embodiments, the bio-ink may include one or more types of cells. In various embodiments, the cells may be autologous cells (i.e., cells of the patient's own body). In various embodiments, the cells may be allogeneic cells (i.e., cells from people other than the patient). In various embodiments, the cells to be used for skin regeneration may include skin cells and stem cells to be differentiated into skin cells. In various embodiments, the skin cells may include keratinocytes, melanocytes, Langerhans cells, and Merkel cells. In various embodiments, stem cells refer to cells with differentiation efficacy, which may include fibroblasts, myoblasts, adult stem cells, mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, neural-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, umbilical cord blood stem cells, or a combination thereof.

[0094] In various embodiments, the bio-ink may include one or more additives that can support cell survival and growth. For example, the bio-ink may include culture medium, physiological salts, buffers, growth factors (i.e., substances that can regulate cell growth and function), differentiation factors (i.e., substances that can induce cell differentiation into tissues or other cells), and combinations thereof. For example, growth factors or differentiation factors may include transforming growth factor (TGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), cytokines, chemokines, or combinations thereof.

[0095] In various embodiments, the bio-ink may be at an acidic pH, a neutral pH, or an alkaline pH. In various embodiments, the bio-ink may be at a physiological pH of about 7.4. In various embodiments, the bio-ink may be used for bio-printing when the temperature falls within a range of about 4°C to about 37°C. For example, the bio-ink, such as acidified collagen, may be kept ice-cold in a box, for example, maintained at about 0°C to about 8°C, typically on ice, and then mixed and deposited ice-cold. When the bio-ink flows out and falls on the skin surface, it will warm to about 37°C. In various embodiments, the bio-ink may be used for bio-printing at room temperature (e.g., from about 20°C to about 30°C). In various embodiments, the bio-ink may be sterile. In various embodiments, the bio-ink may be biocompatible. In various embodiments, the bio-ink may be non-toxic. In various embodiments, the bio-ink may have a viscosity suitable for dispensing from the first chamber of the storage module. In various embodiments, the bio-ink may have a viscosity suitable for being applied to the surface of the patient by a rotatable applicator.

[0096] In various embodiments, the bio-ink can have a viscosity value falling within the range of about 113 cP to about 8471 cP. In various embodiments, the viscosity of the bio-ink can fall within a range having a starting point and an end point selected from the following group of numbers: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, and 8500 cP.

[0097] Crosslinking agent

[0098] In various embodiments, the crosslinking agent may include calcium chloride, calcium sulfate, calcium carbonate, calcium (Ca 2+ ), magnesium (Mg 2+ ), base (such as sodium hydroxide), glutaraldehyde, genipin, nordihydroguaiaretic acid, tannic acid, proanthocyanidins, glycosaminoglycans (GAGs), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), tetra(ethylene glycol) diacrylate (TEGDA), poly(ethylene glycol) diacrylate (PEGDA), and combinations thereof.

[0099] In one example, when the bio-ink is acidified collagen, cross-linking can be initiated by neutralization of an acid. The acidified collagen can be stored in a first chamber of a storage module, and a base such as sodium hydroxide can be stored in a second chamber of the storage module. Mixing the base with the acidified collagen initiates neutralization of the acid and causes the collagen to form a gel.

[0100] In another example, when the bio-ink is alginate, cross-linking can be induced by mixing with a calcium salt such as a calcium chloride solution. The bio-ink containing alginate can be stored in a first chamber of the storage module, and the calcium chloride solution can be stored in a second chamber of the storage module. The mixing of alginate and calcium chloride causes cross-linking to occur, and the alginate forms a gel.

[0101] Methods of making devices for skin bioprinting

[0102] In various embodiments, a method for manufacturing an apparatus for skin bioprinting is provided, the method comprising: providing a dispenser module configured to drive and dispense bio-ink from a first chamber, and to drive and dispense a cross-linking agent from a second chamber; providing a storage module coupled to the dispenser module, the storage module comprising a first chamber for storing the bio-ink and a second chamber for storing the cross-linking agent; providing a mixing module coupled to the storage module for mixing the bio-ink and the cross-linking agent dispensed from the first chamber and the second chamber, respectively; and providing a rotatable applicator coupled to the mixing module for rotatably applying the mixture of the bio-ink and the cross-linking agent from the mixing module to a surface of a patient in need of skin regeneration.

[0103] In various embodiments, the method may further include providing a cooling module configured to include a cooling element for cooling the bio-ink, the cooling being to prevent premature gelation of the bio-ink. In various embodiments, the method may further include arranging the cooling module adjacent to or at least partially surrounding the mixing module, such that, in use, the cooling element included therein is capable of cooling the mixture of the bio-ink and the cross-linking agent.

[0104] In various embodiments, the step of providing a dispenser module may include providing a first piston and a second piston, the first piston being configured to be displaced within a first chamber to dispense bio-ink from the first chamber, and the second piston being configured to be displaced within a second chamber and dispense a crosslinking agent from the second chamber. In various embodiments, the step of providing a dispenser module may further include providing one or more pneumatic inlets configured to introduce gas to drive the first and / or second pistons. In various embodiments, the step of providing a dispenser module may further include providing a plunger coupled to the first and second pistons. In various embodiments, the method may further include providing a pneumatic pump coupled to the one or more pneumatic inlets.

[0105] In various embodiments, the step of providing a rotatable applicator may include providing a ball roller configured to roll along a patient surface in need of skin regeneration. In various embodiments, the step of providing a rotatable applicator may include providing a cylindrical roller configured to roll along a patient surface in need of skin regeneration.

[0106] In various embodiments, the method may further include providing a control unit configured to control the dispensing of the bio-ink and the cross-linking agent from the first chamber and the second chamber to the mixing module, respectively, and further configured to control the mixing of the bio-ink and the cross-linking agent in the mixing module. In various embodiments, the method may further include providing an activation switch electrically coupled to the control unit for activating and / or deactivating the control unit.

[0107] Method of applying a mixture of bio-ink and cross-linking agent

[0108] In various embodiments, a method for applying a mixture of a bio-ink and a cross-linking agent on a surface is provided, the method comprising providing a storage module, wherein the bio-ink is stored in a first chamber and the cross-linking agent is stored in a second chamber; using a dispenser module to drive and dispense the bio-ink from the first chamber to a mixing module, and using a dispenser module to drive and dispense the cross-linking agent from the second chamber to the mixing module; mixing the bio-ink and the cross-linking agent dispensed from the first and second chambers, respectively, in the mixing module; and applying the mixture of the bio-ink and the cross-linking agent on the surface using a rotatable applicator.

[0109] In various embodiments, the surface can be a skin surface of a patient in need of skin regeneration. In various embodiments, the method can be performed at room temperature of about 20° C. to about 30° C. In various embodiments, the method can further include cooling the bio-ink to prevent premature gelation of the bio-ink. In various embodiments, the method can further include rolling a rotatable applicator along the surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1A is a schematic diagram of an apparatus / bioprinter for skin bioprinting in an example embodiment.

[0112] Figure 1B is a schematic diagram of an apparatus for skin bioprinting in an exemplary embodiment.

[0113] Figure 2A is a schematic diagram of an apparatus for skin bioprinting in another example embodiment.

[0114] Figure 2B is a schematic cross-sectional view of an apparatus for skin bioprinting in an example embodiment.

[0115] Figure 3 is a photograph showing static mixing of a simulated bio-ink base and a simulated cross-linker solution using a static mixer in an example embodiment.

[0116] Figure 4 is a photograph of a plunger, such as a rear plunger, in an example embodiment.

[0117] Figure 5 is a photograph of a partially disassembled apparatus / bioprinter for skin bioprinting in an example embodiment.

[0118] Fig. 6A is a photograph showing an apparatus / bioprinter for skin bioprinting in yet another example embodiment.

[0119] Figure 6Bis a photograph of the device / bioprinter showing application of material to a surface in an example embodiment.

[0120] Figure 7 is a schematic diagram showing the composition of the total gel mixture used for bioprinting in an example embodiment.

[0121] Fig. 8A is a photograph of a well plate containing a collagen gel in an example embodiment.

[0122] Figure 8B is a photograph showing a close-up view of the collagen gel in a particular well in an example embodiment.

[0123] Figure 8C is a photograph showing a collagen gel being grasped by a pair of tweezers in an example embodiment.

[0124] Fig. 9 is a graph showing viscosity profiles of different formulations in example embodiments.

[0125] Detailed Description of the Drawings

[0126] The example embodiments of the present disclosure will be better understood and apparent to those of ordinary skill in the art from the following discussion and, if applicable, in conjunction with the accompanying drawings. It should be understood that other modifications related to structural, chemical, electrical, and biological changes may be made without departing from the scope of the present invention. The example embodiments are not necessarily mutually exclusive, as some may be combined with one or more embodiments to form new example embodiments. The example embodiments should not be construed as limiting the scope of the present disclosure.

[0127] Figure 1A is a schematic diagram of an apparatus / bioprinter (100) for skin bioprinting in an example embodiment. Figure 1Bis a cross-sectional view of a schematic diagram of an apparatus (100) for skin bioprinting in an exemplary embodiment. The apparatus (100) for skin bioprinting includes a dispenser module (102) configured to drive and dispense bio-ink from a first chamber (110) and to drive and dispense a cross-linking agent from a second chamber (112); a storage module (104) coupled to the dispenser module (102), the storage module (104) including a first chamber (110) for storing bio-ink and a second chamber (112) for storing a cross-linking agent; a mixing module (106) coupled to the storage module (104) for mixing the bio-ink and the cross-linking agent dispensed from the first and second chambers (110, 112), respectively; and a rotatable applicator, for example, a roller ball applicator (108) coupled to the mixing module (106), rotatably applying the mixture of the bio-ink and the cross-linking agent from the mixing module (106) to the surface of a patient in need of skin regeneration.

[0128] In an exemplary embodiment, the dispenser module (102) further includes a first piston (114) and a second piston (116), the first piston being configured to displace / move within the first chamber (110) to dispense the bio-ink from the first chamber (110), and the second piston being configured to displace / move within the second chamber (112) and dispense the crosslinking agent from the second chamber (112). In an exemplary embodiment, the dispenser module (102) further includes a plunger (118) coupled to the first and second pistons (114, 116). In an exemplary embodiment, the dispenser module (102) further includes a pneumatic inlet (120) configured to introduce gas to displace / move the plunger (118) and the first and second pistons (114, 116). The pneumatic inlet (120) can be coupled to a positive displacement pump, such as a pneumatic pump (not shown), which is configured to apply gas or air pressure to displace / move the plunger (118) and the pistons (114, 116). In an example embodiment, the plunger (118) is configured to move in response to a force exerted thereon by an applied air or gas pressure, which in turn causes the first and second pistons (114, 116) to move in tandem. In an example embodiment, the pressure provided to the pneumatic dispenser module (102) is adjustable to achieve different deposition rates. In an example embodiment, the plunger (118) and the pistons (114, 116) can be made of a material having a relatively low coefficient of friction, such as polytetrafluoroethylene (PTFE), to reduce the force required to displace the plunger (118) and the pistons (114, 116). This can advantageously facilitate fine control of the flow rate / dispensing rate of the bio-ink and the cross-linking agent.

[0129] In an exemplary embodiment, the mixing module (106) includes a mixing chamber (122) having a mixer, such as a static mixer disposed within the mixing chamber. The static mixer may include a series of immovable mixing elements, such as spiral elements and / or baffles, disposed within the mixing chamber (122) to direct flow and promote mixing of the mixture of the bio-ink and the cross-linking agent as it moves from an inlet to an outlet of the mixing module (106). In an exemplary embodiment, the static mixer may enable rapid mixing of the bio-ink and the cross-linking agent.

[0130] In an exemplary embodiment, the design of the ball applicator (108) allows the material to be easily deposited on the contoured body. The use of the ball applicator (108) for depositing the mixture of bio-ink and cross-linking agent is more relevant in the case of a handheld device because the device is allowed to be manipulated on the contoured surface. The physical contact between the ball applicator (108) and the surface of the rolling action helps to spread the mixture of bio-ink and cross-linking agent onto the surface. The inventors have recognized that maintaining contact with the contoured surface with the XYZ Cartesian platform of a conventional 3D printing instrument is technically difficult because the instrument must be programmed to dynamically adjust the position of its print head, which is not easily achieved because most 3D printers using such platforms have pre-installed tool paths and cannot be changed on the fly.

[0131] In an exemplary embodiment, the device (100) further includes a cooling module in the form of a hollow compartment (124) surrounding the mixing module (106). The hollow compartment (124) allows for a cooling element, such as a phase change material, to be contained therein. The cooling element is used to provide cooling / chilling to the mixture of the bio-ink and the cross-linking agent. To prevent clogging of the mixer, the bio-ink, such as collagen, should be kept chilled both before and after neutralization. This can be achieved using a cooling element, such as a phase change cooling gel material (e.g., hydroxyethyl cellulose, sodium polyacrylate, or vinyl coated silicone), which fills a specially designed hollow compartment (124) in the device (100). Prior to use, the device (100) can be stored in a refrigerator to cool the phase change material. After being deposited on the surface of the patient, the bio-ink warms to the patient's body temperature and begins to gel.

[0132] In an exemplary embodiment, the device (100) is a handheld bioprinter designed to deposit bio-ink, such as cell-loaded bio-ink, directly onto a surface, such as a wound bed and a contoured surface, such as the contoured body of a patient. The inventors focused on usability when designing the device so that the device can be deployed intraoperatively without being too cumbersome. Therefore, in an exemplary embodiment, the device (100) has a relatively small size, low instrumentation requirements, and relies on compressed air to dispense materials. For example, the device (100) can be configured to operate with only one pipe connected thereto (to provide air pressure) and a single cable triggered by a finger. The single cable is also optional because the device (100) can be triggered alternately with a foot switch. This makes the device (100) more compact and easy to use and manipulate.

[0133] Figure 2A is a schematic diagram of an apparatus (200) for skin bioprinting in another exemplary embodiment. Figure 2B is a cross-sectional view of a schematic diagram of an apparatus (200) for skin bioprinting in an example embodiment.

[0134] In an exemplary embodiment, the device (200) includes a mixing module (202) including a mixing element, such as a static mixing element (204), disposed within a mixing chamber (206) and configured to mix cells, a bio-ink base, such as collagen, and a cross-linking agent. The device (200) further includes a rotatable applicator, such as a ball (208) coupled to the mixing module (202) and configured to rotatably apply the mixture (210) of the bio-ink base, cells, and cross-linking agent from the mixing module (202) to a surface (212), such as a surface of a patient in need of skin regeneration. The device (200) further includes a cooling module in the form of a thermal control element (214) disposed adjacent to or at least partially surrounding the mixing module (202) so that the thermal control element (214) can cool the mixture (210) of cells, bio-ink, and cross-linking agent when in use.

[0135] In an exemplary embodiment, the surface (212) is a conformable dermal template, i.e., a dressing placed on the surface of a patient's skin. By coupling a mixing mechanism (for mixing a bio-ink base with cells and a cross-linking agent) to a rotatable applicator (e.g., a roller ball fitting), the device (200) can advantageously achieve uniform deposition of the mixture (210) of cells, bio-ink, and cross-linking agent onto the contoured surface (212).

[0136] In an exemplary embodiment, the thermal control element (214) helps prevent premature gelation of the bio-ink, thereby maintaining a longer working time of the bio-ink, allowing clinicians greater flexibility and wiggle room in the operating room. In an exemplary embodiment, by coupling a mixing mechanism (for mixing the bio-ink base, cells, and cross-linking agent) to a ball bearing assembly, the mixture can be uniformly deposited on a contoured surface, such as the human body. By depositing continuous layers of bio-ink loaded with cells, such as keratinocytes, directly onto a surface, such as the dermis, better cosmetic results can be achieved for the patient.

[0137] Figure 3 A photograph showing static mixing of a simulated bio-ink base (300) and a simulated crosslinker solution (302) using a static mixer (304) in an exemplary embodiment. In the exemplary embodiment, for ease of observation, the simulated bio-ink base (300) is a colorless solution and the simulated crosslinker solution (302) is a blue solution. The simulated bio-ink base (300) and the simulated crosslinker solution (302) are simultaneously injected from the corresponding bio-ink storage chamber (306) and the crosslinker storage chamber (308) at one end of the static mixer (304). The mixture of the simulated bio-ink base (300) and the simulated crosslinker solution (302) flows through a series of baffles (e.g., 310) in the static mixer (304) and then flows out through an outlet on the opposite end of the static mixer (304). Experiments have shown that the static mixer is able to achieve rapid and uniform mixing of the simulated bio-ink base (300) and the simulated cross-linking agent solution (302), as evidenced by the uniform color of the mixture discharged at the outlet of the static mixer (304).

[0138] Figure 4 is a photograph of a plunger, such as rear plunger (400), in an exemplary embodiment. Rear plunger (400) is similar in construction to Figure 1B The solution cannot be driven directly with pressurized air due to the difference in viscosity between the bio-ink base, such as collagen base, and the cross-linking agent, because the lower viscosity cross-linking agent will flow much faster than the bio-ink base. In an exemplary embodiment, the bio-ink base and the cross-linking agent are driven using a positive displacement mechanism, such as by using a pneumatic positive displacement dispenser. Such a dispenser may include a piston connected to two (compare Figure 1B 114 and 116) rear plunger (comparison Figure 1B 118).

[0139] In an exemplary embodiment, the rear plunger (400) is a close fitting but low friction plunger that is specially manufactured using a material having a relatively low coefficient of friction, such as machined from a block of PTFE. The rear plunger (400) is able to move freely while maintaining a close fit with the inner surface of the dispenser module. In this way, a relatively low pressure drive piston (compared to Figure 1B 114 and 116), which in turn allows for fine tuning of the dispensing rate. When connected to a bio-ink cartridge, the minimum operating pressure required to drive the rear plunger (400) is about 2 psi to about 4 psi, significantly lower than the rear plunger of a conventional positive displacement dispenser.

[0140] On the other hand, in conventional positive displacement dispensers, the rear plunger is typically sealed with an O-ring, such as a double O-ring, and does not begin to move until a pressure of about 14-16 psi is applied. However, since static friction is higher than dynamic friction, once the rear plunger begins to move, the movement becomes fast unless the viscous material to be dispensed resists movement. Because conventional positive displacement dispensers are designed for high viscosity materials such as two-component epoxy resins, when using materials with relatively low viscosity, such as collagen and culture medium, the dispensing rate is significantly higher and is not easy to control.

[0141] Figure 5 is a photograph of a partially disassembled apparatus / bioprinter (500) for skin bioprinting in an exemplary embodiment. In partially disassembled form, the apparatus (500) is shown to include a dispenser module (502) including a first piston, such as a pneumatic piston (504) and a second piston, such as a pneumatic piston (506) (compare Figure 1B 114 and 116). The pneumatic pistons (504, 506) can be coupled to a plunger (not shown) driven by a pneumatic pump. The device (500) further includes an activation switch, such as a finger-activated trigger (508) placed on an outer surface of the device (500) that is easily accessible to a user's finger. In other words, the dispensing of the bio-ink and cross-linking agent from the device (500) is based on user input. When the trigger (508) is pressed (i.e., activated), the pneumatic pistons (504, 506) are driven to dispense the bio-ink and cross-linking agent, which are then mixed and applied / rolled onto the surface. When the trigger (508) is released (i.e., deactivated), the bio-ink and cross-linking agent are not dispensed, nor do they leak from the device (500).

[0142] Fig. 6A is a photograph showing an apparatus / bioprinter (600) for skin bioprinting in yet another example embodiment. Figure 6B is a photograph of a device / bioprinter (600) showing application of material to a surface (614) in an example embodiment.

[0143] In an exemplary embodiment, the device (600) includes a first box (602) storing a bio-ink and a second box (604) storing a cross-linking agent. The device (600) further includes a mixing module (606) for mixing the bio-ink and the cross-linking agent dispensed from the first box (602) and the second box (604). The device (600) further includes a rotatable applicator, such as a ball (608), for applying the mixture (612) of the bio-ink and the cross-linking agent dispensed from the mixing module (606). The ball (608) is connected to the mixing module (606) via a ball fitting interface (610), which is configured to connect the nozzle of the mixing module (606) to the ball (608) and allow the mixed bio-ink (612) to be uniformly and quickly deposited on the surface (614). The device (600) further includes a cooling module (616), which includes a hollow compartment containing a cooling element for cooling the mixture (612) of the bio-ink and the cross-linking agent. The device (600) additionally includes an activation switch, such as a finger trigger (618) disposed on an outer surface of the device (600).

[0144] In an exemplary embodiment, the device (600) is a 3D printed prototype made of polylactic acid (PLA) plastic. Figure 1A and Figure 1B A simplified version of the device (100) in FIG. 6 is shown. Since plastic is not a good conductor of heat, a cooling / icing element is not included in the cooling module (616). It will be appreciated that the device can be made using other types of materials such as aluminum. It will also be appreciated that the prototype can be improved based on better ergonomics to obtain a better feel in the hand.

[0145] During operation, the coupler module (compare Figure 1B The pneumatic pump (102) of the device (600) is set to operate at 4 psi and is activated by a finger trigger (618). Upon activation, the bio-ink and cross-linking agent are dispensed from the respective first and second cartridges (602, 604) into the mixing module (606), and the mixture (612) of the bio-ink and cross-linking agent is then dispensed from the nozzle of the mixing module (606) to the ball applicator (608). The ball applicator (608) contacts the surface (614) and rolls along the surface. The rolling action of the ball applicator evenly distributes the mixture (612) of the bio-ink and cross-linking agent on the surface (614). Upon deactivation or release of the finger trigger (618), the dispensing of the bio-ink and cross-linking agent stops and the device (600) does not leak. In an exemplary embodiment, the device (600) is capable of applying the mixture (612) of the bio-ink and cross-linking agent to a surface of approximately 100 cm in approximately 10 seconds. 2 on the area.

[0146] Figure 7is a schematic diagram showing the composition of the total gel mixture for bioprinting in an example embodiment. One third of the total gel mixture volume consists of 1x culture medium (with or without cells). The other two thirds of the total gel mixture volume consists of 10% volume of 10x culture medium and 90% type I collagen solution, double distilled water, and sodium hydroxide.

[0147] In addition to nutrient-rich culture media and cells, bio-inks contain a base and a cross-linking agent. After mixing, the bio-ink forms a hydrogel after a period of time. Depending on the base material, the gelation can occur almost immediately (e.g., alginate plus calcium chloride cross-linking agent solution), or after about 15 minutes (e.g., at 37°C, 0.15% neutralized collagen).

[0148] Collagen is the main component of the extracellular matrix of tissues and can be derived from pig, cow or fish tissues. Typically, collagen can be dissolved in acid. After mixing with an appropriate amount of alkali to neutralize the acid, collagen forms a gel. The gelation process can be accelerated by incubation at 37°C, or slowed down by ice cooling. Table 1 below shows the time it takes for fish collagen of different concentrations to form a gel. Based on the time it takes for gelation to occur, sufficient time should be allocated for all culture media / cells to be prepared before they reach the patient.

[0149] Table 1: Curing time of fish collagen at different concentrations

[0150] Fish collagen % 0.5 0.4 0.2 0.15 Curing time (mins) 10 10 12.5 15

[0151] Fig. 8A is a photograph of a well plate containing collagen gel in an example embodiment. Figure 8B is a photograph showing a close-up view of the collagen gel in a particular well in an example embodiment. Figure 8C is a photograph showing a collagen gel held by a pair of tweezers in an exemplary embodiment.

[0152] Fig. 9 is a graph showing viscosity curves of different formulations in an exemplary embodiment. Alginate has a similar viscosity curve to 0.33% fish collagen and can therefore be used as a substitute for fish collagen when testing the device for skin bioprinting disclosed herein.

[0153] application

[0154] Embodiments of the disclosure provided herein provide devices for skin bioprinting, methods of making devices for skin bioprinting, and methods of applying a mixture of a bio-ink and a crosslinking agent. Embodiments of the bioprinting device include a dispenser module, a storage module, a mixing module, a rotatable applicator, and an optional cooling module.

[0155] Advantageously, the modularity of the device can allow individual components to be replaced independently of one another. This can advantageously minimize cross-contamination when reusing the device, since components that come into contact with biological matter (e.g., a patient's cells) can be disposed of and replaced after use. Furthermore, the relatively simple construction of the device can facilitate easy replacement of its various components, compared to more complex bioprinting devices known in the art (whose components are clearly not disposable due to their complex structures).

[0156] Advantageously, the device is handheld and can be easily maneuvered over contoured surfaces (e.g., surfaces of human anatomy). In particular, the use of pneumatic inlets (rather than multiple control cables for motors and coolant lines in conventional bioprinters) can help reduce the size of the device, making it smaller and easier to handle.

[0157] Even more advantageously, an apparatus for skin bioprinting can be capable of depositing a substantially continuous layer of bio-ink (e.g., bio-ink loaded with keratinocytes) directly onto a surface (e.g., dermis) of a patient, thereby achieving a better cosmetic effect for the patient. In particular, the use of a roller ball can advantageously allow a relatively large surface area to be covered in a relatively short time compared to a nozzle for dispensing a material (e.g., a mixture of bio-ink and a cross-linking agent). The roller ball can advantageously allow material to be more easily deposited on contoured surfaces (e.g., contoured body surfaces such as the nose and ears).

[0158] Those skilled in the art will appreciate that other changes and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, merged, adopted, modified, included, etc. between different exemplary embodiments. Therefore, the embodiments herein should be considered in all respects as illustrative and not restrictive.

Claims

1. A device for skin bioprinting, comprising: a dispenser module configured to drive and dispense the bio-ink from the first chamber and to drive and dispense the cross-linking agent from the second chamber; a storage module coupled to the dispenser module, the storage module comprising a first chamber for storing the bio-ink and a second chamber for storing the cross-linking agent; a mixing module coupled to the storage module, the mixing module for mixing the bio-ink and the cross-linking agent dispensed from the first and second chambers, respectively; and A rotatable applicator is coupled to the mixing module, the rotatable applicator rotatably applying the mixture of the bio-ink and the cross-linking agent from the mixing module to a surface of a patient in need of skin regeneration.

2. The device according to claim 1 further comprises a cooling module, wherein the cooling module is configured to include a cooling element for cooling the bio-ink, wherein the cooling is to prevent premature gelation of the bio-ink.

3. An apparatus according to claim 2, wherein the cooling module is arranged adjacent to or at least partially surrounding the mixing module so that, when in use, the cooling element contained therein is capable of cooling the mixture of the bio-ink and the cross-linking agent.

4. The apparatus of claim 1 , wherein the dispenser module comprises a first piston and a second piston, wherein the first piston is configured to be displaced within a first chamber to dispense bio-ink from the first chamber, and the second piston is configured to be displaced within a second chamber and dispense a cross-linking agent from the second chamber.

5. The device according to claim 4, wherein the distributor module further comprises one or more pneumatic inlets configured to introduce gas for driving the first and / or second piston.

6. The device of claim 4, wherein the dispenser module further comprises a plunger coupled to the first and second pistons.

7. The apparatus of claim 1, wherein the mixing module comprises a static mixer.

8. The device of claim 1, wherein the rotatable applicator comprises a roller ball configured to roll along a surface of a patient in need of skin regeneration.

9. The device of claim 1, wherein the rotatable applicator comprises a cylindrical roller configured to roll along a surface of a patient in need of skin regeneration.

10. The apparatus according to claim 1, further comprising a control unit configured to control the distribution of the bio-ink and the cross-linking agent from the first chamber and the second chamber to the mixing module, respectively.

11. The apparatus according to claim 10, wherein the control unit is further configured to control mixing of the bio-ink and the cross-linking agent in the mixing module.

12. The device of claim 10, wherein the device further comprises an activation switch electrically coupled to the control unit for activating and / or deactivating the control unit.

13. The device of claim 5, wherein the device further comprises a pneumatic pump connected to the one or more pneumatic inlets.

14. The device of claim 1, wherein the device is a handheld device.

15. The device of claim 1, wherein the storage module is removably coupled to the dispenser assembly.

16. The apparatus of claim 1, wherein the mixing module is removably coupled to a storage module.

17. The device of claim 1, wherein the first chamber and the second chamber of the storage module are independently detachable.

18. The apparatus of claim 2, wherein the cooling module comprises a cooling element.

19. The device of claim 18, wherein the cooling element comprises a phase change material.

20. A method of making a device for skin bioprinting, the method comprising: providing a dispenser module configured to drive and dispense the bio-ink from the first chamber and to drive and dispense the cross-linking agent from the second chamber; providing a storage module coupled to the dispenser module, the storage module comprising a first chamber for storing the bio-ink and a second chamber for storing the cross-linking agent; providing a mixing module coupled to the storage module, the mixing module mixing the bio-ink and the cross-linking agent dispensed from the first and second chambers, respectively; and A rotatable applicator coupled to the mixing module is provided, the rotatable applicator rotatably applying the mixture of the bio-ink and the cross-linking agent from the mixing module to a surface of a patient in need of skin regeneration.

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