System for providing a bioink

Abstract

The present invention relates to a system for providing a bioink. The system comprises: a) at least two spray configured to receive at least two syringes comprising a nozzle each, each syringe configured to receive one or more components of the bioink; b) a housing, configured to comprise the at least two spray heads and comprising at least two housing nozzles, the housing nozzle comprising a diameter bigger than the diameter of the spray head nozzle; c) a gas inlet connected to the housing configured to receive a gas; and d) a control system.

Description

[0001] SYSTEM FOR PROVIDING A BIOINK

[0002] TECHNICAL FIELD

[0003] The present invention belongs to the field of medical devices. Particularly, the present invention relates to a system for providing a bioink in the form of a spray or by extrusion, and to a method of providing a bioink for the treatment of tissue injuries or damages using a system thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Skin is one of the most important defense mechanisms in the body, protecting it against pathogens and chemical and physical hazards. The skin is composed of three main layers: the top layer is the epidermis, the middle and thicker layer is the dermis, and the subcutaneous tissue is the hypodermis. Skin wounds or injuries are breaks in the skin tissue, which may result from different causes, such as physical or chemical traumas (including burns or removal of skin during surgery), genetic irregularities, or skin diseases, such as epidermolysis bullosa, perianal fistulae or chronic wounds (like diabetic foot ulcers, pressure ulcers, and leg ulcers). Shallow injuries can usually be regenerated naturally by the skin's self-healing functions, but in deep wounds, the skin's regenerative components are destroyed, therefore requiring therapeutic interventions to facilitate wound healing.

[0006] Traditionally, the gold standard in clinical practice for treating deep wounds has been the use of autologous skin grafts (autografts). Despite their effectiveness, autografts present important limitations, such as the unavailability of sufficient donor skin in cases like major burn patients, and the creation of a secondary wound for the patient when harvesting said donor skin. Other grafting options include the culture of cells harvested from a small donor site to amplify their number, obtaining cultured epithelial autografts (CEAs), which require weeks to become available, or the temporary use of allografts or xenografts, which have the risk of diseases transmission and immune rejection.

[0007] On the other hand, thanks to the advances in regenerative medicine and tissue engineering, different biofabrication methods (such as 3D bioprinting or electrospinning) have been developed for the production of skin substitutes, which are defined as a heterogeneous group of substances (including hydrogels, cell suspensions, films, 3D scaffolds or cell sheets) intended to cover the wound and act as a barrier to avoid infections and fluid loss, promote wound healing, and reduce pain. Numerous cellular and acellular skin substitutes are commercially available for clinical use. The biofabrication processes generally make use of bioinks, either acellular formulations containing biologically active components or molecules (but that can be subsequently seeded with cells afterward), or cell-based formulations that may also contain biomaterials and other biologically active components. To manufacture skin substitutes, bioinks have been formulated with various natural and synthetic polymeric biomaterials, that assemble into three-dimensional networks with a structure similar to the natural ECM, where cells can adhere, proliferate, differentiate, and promote skin regeneration. These biomaterials ought to have good mechanical properties, be biocompatible and biodegradable, be capable of maintaining humidity, and have enough porosity to allow cell migration and proliferation and the transport of nutrients and metabolic wastes. Some examples of biomaterials used for skin substitutes are collagen, fibrin, alginate, gelatin, glycosaminoglycans (GAGs), or chitosan.

[0008] One biofabrication technique disclosed in the prior art for application in skin regeneration is the use of skin sprays, which present several advantages for the delivery of bioinks or biomaterial inks to the wound bed: the possibility to treat large wounds or areas with unfavorable topography, in a rapid manner, with much more facility, and obtaining a homogeneous distribution of the sprayed material.

[0009] Acellular sprays generally consist of biomaterial inks that, when sprayed over the wound bed, create thin films or dressings covering the wounds, protecting them against TEWL and infections, and promoting the wound healing process. There are several acellular spray products that have been commercialized for decades now, the majority of them based on fibrin due to its hemostatic properties: for example, TISSEEL® and ARTISS® by Baxter [1 ,2], VISTASEAL™ by Johnson & Johnson [3], or Vivostat's Fibrin Sealant [4], Other biomaterials that have been studied for acellular sprays are alginate [5], gelatin [6], chitosan [7], hyaluronic acid (HA) [8], and peptin [9],

[0010] On the other hand, cellular sprays apply different types of cell suspensions or bioinks. For in vivo studies, autologous epidermal cells (normally fibroblasts or keratinocytes) are mainly used, although allogeneic cells have also been studied. Once sprayed over the wound bed, epidermal cells can remain viable and proliferate, promoting re- epithelialization. Cell spray autografting consists in taking a biopsy of the patient's undamaged skin, digesting said biopsy, and obtaining a solution of autologous epidermal cells that can be sprayed immediately over the wound.

[0011] In contrast to traditional sheet autografts, cell sprays only require a much smaller donor area and offer a cost-effective, simple, and immediate treatment for the patient. Moreover, the harvested autologous cell can also be cultured to expand their number before being sprayed. There are many reports of cellular sprays being studied in v / fro[10,11], in animals [12, 13] and in patients [14-17], for burns and wound healing applications, typically using airbrushes or syringes with spray nozzles as the spraying devices. However, few cell spray products have been approved for commercialization and use in clinical practice. The ReCell® kit by Avita Medical was the first "spray-on-skin" treatment approved by the U.S. Food and Drug Administration (FDA) in 2018, and it employs an enzyme solution to isolate autologous epidermal cells from a small biopsy of the patient's healthy skin, which are then suspended in a lactate solution and sprayed over the wound using a syringe with a spray nozzle

[0018] , Similarly, RenovaCare developed another enzymatic isolation technique to obtain a solution of autologous epidermal cells from a biopsy of the patient, called the "CellMist Solution", which is delivered through their SkinGun, an electronically controlled pneumatic spray device. However, this product has not received approval for commercialization yet

[0019] ,

[0012] The use of a decellularized skin extracellular matrix (dsECM) in fibrinogen hydrogels and its benefits for skin bioprinting applications has been described previously

[0020] , The decellularized extracellular matrix (dECM) is prepared from cells by decellularization, i.e. removal of cellular components, and thus retains the original ECM structural and functional properties such as nanostructure, biochemical complexity, and bio inductive properties. The full composition of such dECM therefore varies depending on the source. dECM can be obtained from cultured cells, which has lower batch-to-batch variability but requires extremely high cell numbers and extended culture times, resulting in low yields. This makes large-scale production costly and inefficient. It can also be obtained from decellularized tissue which allows for higher yields, but the availability depends on donor tissue sources. Additionally, its composition and properties can vary significantly depending on patient characteristics, tissue origin, and processing methods, potentially affecting reproducibility and performance. Additional drawbacks of dECMs are their complex processing (cell removal while preserving ECM components is challenging and can lead to batch inconsistencies) and their biochemical variability (composition of dECM is not standardized and may vary between preparations). Furthermore, they face regulatory challenges, since dECM originates from human or animal tissue, its use in clinical applications may require additional regulatory approvals compared to defined biomaterials.

[0013] Normally, cell suspensions can run off when sprayed over the body's convex structures, reducing the number of cells that stay in contact with the wound bed. To avoid this, cell suspensions can be sprayed in combination with biomaterials, such as fibrin, to ensure that the solution stays in place and cells adhere to the wound.

[0014] For example, US2006172008 A1 discloses porous freeze-dried fibrin matrices for in vitro and in vivo cell growth and tissue regeneration in which fibrin and hyaluronic acid are used to prepare the matrix.

[0015] US2017304600A1 discloses a device and methods suitable for producing a cellular spray of cells for covering and growing cells on a damaged tissue, such as a skin wound. The cellular spray comprises tissue regenerating cells in a physiological solution with electrolytes which can be applied to a damaged tissue.

[0016] As mentioned above, despite their advantages, the translation of cell sprays to clinical practice is still limited and very few products have been approved for clinical use to date. There is therefore a need for providing novel spray device solutions that can be used in clinical practice that allows a simpler and easier to use way of providing bioinks at the will of the clinician according to the necessities of the tissue receiving the bioink spray.

[0017] SUMMARY OF THE INVENTION

[0018] A first aspect of the invention relates to a system for providing a bioink in the form of a spray. The system comprises: a) at least two spray heads, each a spray head configured to receive a syringe, each syringe configured to receive one or more components of the bioink and wherein each a spray head comprises a nozzle, b) a housing (20), configured to comprise the at least two spray heads (12a, 12b), and comprising at least two housing nozzles (22a, 22b), each housing nozzle (22a, 22b) associated to a spray head (12a, 12b) and disposed around the spray head nozzle (125a, 125b), the housing nozzle (22a, 22b) comprising a diameter bigger than the diameter of the spray head nozzle (125a, 125b), c) a gas inlet (30) connected to the housing (20) configured to receive a gas (300), and d) a control system (40) comprising at least two valves (42a, 42b), each valve (42a, 42b) associated to a housing nozzle (22a, 22b) and configured to control the flow of the gas (300) through each housing nozzle (22a, 22b). In this first aspect of the invention, each valve is further configured to control the provision of the one or more components of the bioink from each syringe through its spray head nozzle, by controlling the flow of gas through its housing nozzle, and the at least two valves are configured to provide the one or more components of the bioink of their respective syringe independently. Moreover, each spray head nozzle is completely contained within its housing nozzle.

[0019] In a preferred embodiment of the first aspect of the invention, the at least two valves are configured to provide the one or more components of the bioink of their respective syringe sequentially and / or simultaneously.

[0020] In another preferred embodiment of the first aspect of the invention, each spray head nozzle is completely contained within its housing nozzle. More preferably, it is completely contained within its housing nozzle such that the provision of the one or more components of the bioink from each syringe is controlled through a venturi effect on the spray head nozzle through the flowing of gas through its housing nozzle.

[0021] In another preferred embodiment of the first aspect of the invention, each of the at least two valves is an electric valve and controlled through an actuator on the housing surface.

[0022] In another preferred embodiment of the first aspect of the invention, at least one of the at least two spray head nozzles comprises a diameter of 200pm and its associated housing nozzle comprises a diameter of 500 pm.

[0023] In another preferred embodiment of the first aspect of the invention, the at least two syringes are disposable syringes.

[0024] In another preferred embodiment of the first aspect of the invention, the housing comprises Acrylonitrile Butadiene Styrene (ABS).

[0025] In another preferred embodiment of the first aspect of the invention, the system further comprises a manometer to regulate the pressure the gas is provided through the gas inlet to the system.

[0026] In another preferred embodiment of the first aspect of the invention, the gas is provided with a pressure between 10 and 20 psi, preferably between 12 and 18 psi, more preferably 15 psi. In another preferred embodiment of the first aspect of the invention, the system further comprises a filter between the gas inlet and the gas, preferably a membrane filter, more preferably a membrane filter of at most 0.22mm.

[0027] In another preferred embodiment of the first aspect of the invention, the viscosity of the bioink is comprised within 0.5 mPa s and 104mPa s.

[0028] In another preferred embodiment of the first aspect of the invention, the bioink is a bioink formulation comprising fibrinogen and a glycosaminoglycans (GAGs) / collagen (Col) matrix, wherein the GAGs / Col matrix comprises at least a) 60 - 75% (w / w) hyaluronic acid, and b) 5 - 20% (w / w) sulphated GAGs, and c) 1 - 5% (w / w) of collagen.

[0029] In a more preferred embodiment, the bioink formulation further comprises human cells, preferably human mesenchymal stem cells (hMSCs), keratinocytes and / or human dermal fibroblasts (hDFs).

[0030] In another more preferred embodiment, the amount of fibrinogen in the bioink formulation is:

[0031] (i) between 1 and 100 mg / ml, preferably about 10 mg / ml, and / or

[0032] (ii) between 0.1 and 10 % (w / v), preferably about 1% (w / v).

[0033] In another more preferred embodiment, the amount of GAGs / Col matrix in the bioink formulation is

[0034] (i) between 1 and 25 mg / ml, and / or

[0035] (ii) between 0.1 and 2.5% (w / v).

[0036] In a particular preferred embodiment of the first aspect of the invention, the control system 40 further comprises the features according to the second aspect of the invention.

[0037] In another preferred embodiment of the first aspect of the invention, the system 100 is configured to provide a bioink 200 comprising a shear rate comprised between 0.1 and 15.000 s-1and a viscosity comprised between 1 and 300.000 mPa s at a shear rate of 1000 s-1; at a pressure comprised between 13.8 and 241.3 kPa.

[0038] In another more preferred embodiment when the system 100 is for providing a bioink 200 in the form of a spray, the system 100 is configured to provide a bioink 200 comprising a shear rate comprised between 1 and 15,000 s1and a viscosity comprised between 1 and 300,000 mPa-s at a shear rate of 1 ,000 s-1; at a pressure comprised between 13.8 and 241.3 kPa.

[0039] In a more preferred embodiment, when the system 100 is for providing a bioink 200 by extrusion, the system 100 is configured to provide a bioink 200 comprising a shear rate comprised between 0.1 and 500 s-1and a viscosity comprised between 1 and 300,000 mPa s at a shear rate of 1 ,000 s-1; at a pressure comprised between 13.8 and 241 .3 kPa.

[0040] A second aspect of the invention refers to a system for providing a bioink by extrusion. The system comprises: a) at least two spray heads, each a spray head configured to receive a syringe, each syringe configured to receive one or more components of the bioink and wherein each a spray head comprises a nozzle, b) a housing, configured to comprise the at least two spray heads, and comprising at least two housing nozzles, each housing nozzle associated to a spray head (12a, 12b) and disposed around the spray head nozzle, the housing nozzle comprising a diameter bigger than the diameter of the spray head nozzle, c) a gas inlet (30) connected to the housing configured to receive a gas, and d) a control system com comprises at least two actuators each actuator (43a, 43b) associated to a housing nozzle, wherein each actuator is configured to control the provision of the one or more components of the bioink from each syringe through its housing nozzle by extrusion; and wherein the at least two actuators are configured to provide the one or more components of the bioink of their respective syringe independently.

[0041] It is noted that all of the preferred embodiments of the first aspect of the invention also apply mutatis mutandis to the second aspect of the invention.

[0042] A third aspect of the invention relates to a method of providing a bioink for the treatment of tissue injuries or damages. The method comprises: providing at least two syringes (10a, 10b) each syringe (10a, 10b) comprising one or more components of the bioink (200) in system (100) according to any one of the embodiments of the first aspect of the invention, and actuating one or more of the at least two valves (42a, 42b) sequentially and / or simultaneously to provide the bioink (200) in the form of a spray over the tissue to be treated and / or actuating one or more of the at least two actuators (43a, 43b) sequentially and / or simultaneously to provide the bioink (200) by extrusion over the tissue to be treated.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] To enable a better understanding of the present disclosure, and to show how the present disclosure may be carried out, reference will now be made, by way of example only, to the accompanying schematic drawings, wherein:

[0045] Figure 1 shows a diagram of a system for providing a bioink in the form of a spray according to one or more embodiments of the invention.

[0046] Figure 2 shows a diagram of part of a system for providing a bioink in the form of a spray according to one or more embodiments of the invention.

[0047] Figure 3 shows an image of the at least two housing nozzles and at least two spray head nozzles and a detail of said nozzles according to one or more embodiments of the invention.

[0048] Figure 4 shows an image of a system for providing a bioink in the form of a spray according to one or more embodiments of the invention.

[0049] Figure 5 shows a diagram of a method of of providing a bioink for the treatment of tissue injuries or damages according to one or more embodiments of the invention.

[0050] Figure 6 shows the Metabolic activity, measured as fluorescence intensity (AU), of sprayed hDFs in FibD hydrogels at days 1 , 2, 3, 5, and 7. Statistical significance: *p < 0.05.

[0051] Figure 7 shows a graphs for the physicochemical, rheological and mechanical characterization of a bioink according to one or more embodiments of the invention. (A) swelling and (B) degradation ratio of Fib and FibD hydrogels. (C) Viscosity of distilled water, 0.9% NaCI, and Fib and FibD inks. (D) Young's Modulus; and Viscoelastic moduli (E, Storage Modulus; F, Loss Modulus), of mice skin and hydrogels. Statistical significance: *p < 0.05; **p < 0.01 ; ***p < 0.005.

[0052] Figure 8 shows a diagram of another system for providing a bioink according to one or more embodiments of the invention.

[0053] Figure 9 shows a plurality of image of a system for providing a bioink according to one or more embodiments of the invention, including (a) an overview of the system, (b) a multifunctional adapter of the device, (c) a spraying head, (d) an extrusion head, (e) and a panoramic view of the device for both printing mechanisms. Fig. 10 shows a set of proof of concepts for constructs from a bioink provided with a device according to one or more embodiments of the invention.

[0054] Fig. 11 shows the rheological and mechanical characterization of different bioinks, (a) Viscosity of distilled water, Fib, Fib-Der, and Fib-Der-TA bioinks, (b) Viscosity of HA, AHA, Alg-Gel-Xan, Alg-Gel-Xan-Der, Alg-Gel-Xan-Chon, and F-F-A bioinks, (c) Young’s Modulus and viscoelastic moduli ((d) Storage Modulus and (e) Loss Modulus) of human skin and hydrogels.

[0055] Fig. 12 shows the cell viability and metabolic activity of hDFs in the sprayed hydrogels, (a) hDFs metabolic activity in Fib (10 mg / mL), Der (1 , 2.5, and 5 mg / mL), and TA (1 mg / mL) bioink. It was measured as fluorescence intensity (AU) after 1 , 2, 3, and 4 days of culture, and all hydrogels were manually fabricated, (b) Sprayed hDFs metabolic activity in Fib (10 mg / mL), Der (2,5 mg / mL), and TA (1 mg / mL) bioink. It was measured as fluorescence intensity (AU) after 1 , 2, 3, and 7 days of culture, and different spraying pressures were evaluated, (c) Cell viability (%) at days 1 and 7. (d) Representative confocal images at days 1 and 7, with live cells stained in green (calcein AM) and dead cells stained in red (ethidium homodimer I; EthD-l). Statistical significance: *p < 0.05, **p < 0.01 , ***p <0.005.

[0056] Fig. 13 shows the cell viability and metabolic activity of hDFs in the extruded hydrogels, (a) hDFs metabolic activity in F-F-N 2%, F-F-N 2% + HA 5%, and F-F-N 2% + AHA 5% bioinks. It was measured as fluorescence intensity (AU) after 1 , 2, 3, 4, and 5 days of culture, and all hydrogels were manually fabricated, (b) Cell viability (%) at days 1 and 6. (c) Representative confocal images at days 1 and 6, with live cells stained in green (calcein AM) and dead cells stained in red (EthD-l). Statistical significance: *p < 0.05, **p < 0.01 , ***p < 0.005.

[0057] Fig. 14 shows the cell viability and metabolic activity of hDFs and hMSCs in the extruded hydrogels, (a) hDFs and hMSCs metabolic activity in Alg (80 mg / mL), Gel (50 mg / mL), Xan (1 mg / mL) bioink, alone or in combination with Der (1 mg / mL) or Chon (10 mg / mL). It was measured as fluorescence intensity (AU) after 1 , 2, 3, 4, and 5 days of culture, and all hydrogels were manually fabricated, (b) Cell viability (%) at days 1 and 6. (c) Representative confocal images at days 1 and 7, with live cells stained in green (calcein AM) and dead cells stained in red (EthD-l). Statistical significance: *p < 0.05, **p < 0.01 , ***p < 0.005. DEFINITIONS

[0058] It must be noted that, as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0059] It is noted that the term “about”, as used herein, refers to + / - 30%, preferably + / - 20%, preferably + / - 15%, more preferably + / - 10%, of the indicated referred value.

[0060] As used herein, the conjunctive term "and / or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and / or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or."

[0061] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". Any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the present invention may be substituted with the term "consisting of', though less preferred.

[0062] When used herein "consisting of' excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0063] As used herein, the term "glycosaminoglycan" or "GAG" refers to a long, unbranched, polysaccharide molecules found on the cell surface or extracellular matrix. Non-limiting examples of glycosaminoglycan include heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, crosslinked or non-crosslinked hyaluronic acid, hexuronyl hexosaminoglycan sulfate, and inositol hexasulfate. Derivatives, salts and mimetics of the above, including low molecular weight heparin are intended to be included in the invention.

[0064] The terms “treatment” and “therapy”, as used in the present application, refer to a set of hygienic, pharmacological, surgical and / or physical means used with the intent to cure and / or alleviate a disease and / or symptoms with the goal of remediating the health problem. The terms “treatment” and “therapy” include preventive and curative methods, since both are directed to the maintenance and / or reestablishment of the health of an individual or animal. Regardless of the origin of the symptoms, disease and disability, the administration of a suitable medicament to alleviate and / or cure a health problem should be interpreted as a form of treatment or therapy within the context of this application. Preferably, the term “treatment” refers to the application or administration of a pharmaceutical composition to a subject who has a disease or condition characterized by ER stress, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward a disease.

[0065] The terms “individual”, “patient” or “subject” are used interchangeably in the present application and are not meant to be limiting in any way. The “individual”, “patient” or “subject” can be of any age, sex and physical condition.

[0066] DETAILED DESCRIPTION

[0067] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0068] A first aspect of the invention relates to a system for providing a bioink in the form of a spray.

[0069] The term “bioink” refers to a biomaterial formulated to mimic the characteristics of natural tissues. Based on its composition, bioinks can be classified into two primary types:

[0070] 1) Cellular Bioinks or simply “bioinks”, which refer to cell-based formulations that may also contain biomaterials and other biologically active components. 2) Acellular Bioinks or biomaterial inks: which refer to acellular formulations containing biologically active components or molecules (but that can be subsequently seeded with cells afterwards). .

[0071] The present invention is thus not limited to any particular type of bioink, and particularly, no discrimination is made between bioinks comprising cells and those not comprising cells, as both fall within the present definition of bioinks. Moreover, in the context of the present invention, the term bioink also comprises any bioink as above-defined in a hydrogel format.

[0072] The term “spray” refers to the dispersion of a substance in the form of fine liquid droplets, propelled under pressure or by other means into the air or onto a surface. The droplets are usually fine and are uniformly distributed over the target area, allowing for even coverage or distribution of the sprayed substance.

[0073] As shown with reference to Fig. 1 the system comprises at least two spray heads 12a, 12b each spray head (12a, 12b) configured to receive a syringe 10a, 10b, each syringe 10a, 10b configured to receive one or more components of the bioink 200 and wherein each a spray head (12a, 12b) comprises a nozzle 125a, 125b.

[0074] The term “syringe” refers to a device used to inject or withdraw fluids. It typically comprises a cylindrical barrel and a plunger. When the plunger is depressed, the fluid in the barrel is expelled through a tip or attached needle. The one or more syringes may be equal between them or may take different sizes, shapes, materials, for example, adapted to one or more components. For example, a syringe 10a may be bigger to accommodate a bigger volume when the one or more components its configured to receive is required in bigger quantities for the bioink the system 100 is configured to provide.

[0075] The term “spray head” refers to a device designed to disperse liquids, often in the form of fine droplets. Its primary function is to control the direction, volume, and characteristics of the spray.

[0076] The term “nozzle” refers to an opening designed to control the direction or characteristics of a fluid flow as it exits an enclosed chamber or pipe.

[0077] Each syringe 10a, 10b is configured to receive one or more components of the bioink (200), such that it is configured to accommodate or integrate the one or more components, but it does not necessarily mean that the one or more components are included within the syringe. The one or more components of the bioink (200), may in turn be also a bioink. Thus, each syringe 10a, 10b may be configured to receive a bioink (200’a, 200’b), wherein the different bioinks (200’a, 200’b), may in turn be components of the final bioink (200) to be provided.

[0078] While spray heads can vary significantly in design and function, a defining feature is the presence of a nozzle 125a, 125b. This nozzle125a, 125b is what allows the spray head to effectively distribute the one or more components of the bioink in the form of a spray.

[0079] The spray head 12a, 12b may be configured to receive a syringe 10a, 10b different ways. This is represented through by arrows 102a and 102b. For example, the syringe 10a, 10b, may be directly attached, such that the syringe's tip or outlet might directly fit into the spray head, ensuring a seamless flow of fluid. Alternatively, the syringe and spray head may connect using a specially designed adapter or coupling that bridges the two, ensuring compatibility, such as quick-connect mechanisms or magnets. Also, a flexible hose or tube might connect the syringe to the spray head, allowing for distance or manoeuvrability between the two components. Another alternative involves.

[0080] The system 100 further comprises a housing 20, configured to comprise the at least two spray heads 12a, 12b, and comprising at least two housing nozzles 22a, 22b, each housing nozzle 22a, 22b associated to a spray head 12a, 12b and disposed around the spray head nozzle 125a, 125b, the housing nozzle 22a, 22b comprising a diameter bigger than the diameter of the spray head nozzle 125a, 125b.

[0081] The nozzle diameter is measured as the size of the aperture on the surface configured to exert the bioink. When the nozzle is circular, the diameter of the nozzle is the diameter of the circumference of the nozzle. When the nozzle is not circular, and takes other opening geometry, the diameter of the nozzle is the diameter of a circular nozzle with the similar hydrodynamic properties.

[0082] Each housing nozzle 22a, 22b is disposed around the spray head nozzle 125a, 125b such that the housing nozzle 22a, 22b and the spray head nozzle 125a, 125b define two nozzles, one within the other. In a preferred embodiment, the housing nozzle 22a, 22b and the spray head nozzle 125a, 125b are located in the same fluid provision axis.

[0083] The system 100, also comprises a gas inlet 30 connected to the housing 20 and configured to receive a gas 300. The gas inlet 30 serves as a crucial component for facilitating the entry of gas 300 into the system 100. This gas inlet 30 can be embodied through a variety of gas connection systems designed to ensure secure and efficient transfer of the gas 300, such as, threaded connectors, quick-connect / disconnect couplings, compression fittings, flange connectors, barbed connectors, bayonet connectors and magnetic couplings. The person skilled in the art may envisage many ways in which the gas inlet may be embodied to receive a gas. In some embodiments, the gas inlet 30 may comprise more than one physical inlet, for example, an inlet per housing nozzle, such that each gas inlet is configured to receive the gas associated to each housing nozzle.

[0084] The term "gas" within the context of the present disclosure is broad and encompasses any type of gaseous substance. This allows for flexibility in applications and ensures compatibility with a wide range of operations and requirements. In a preferred embodiment of the present disclosure, the gas 300 specified is air.

[0085] The system 100, further comprises a control system 40 comprising at least two valves 42a, 42b, each valve 42a, 42b associated to a housing nozzle 22a, 22b. Each valve 42a, 42b is configured to control the flow of the gas 300 through each housing nozzle 22a, 22b.

[0086] The control system 40, although represented as a rectangle in Fig. 1 , is a sophisticated assembly designed to manage the system operation. It can integrate a myriad of components, spanning across the electronic, electric, and pneumatic domains. The skilled person may envisage many ways in which electronic, electric, and / or pneumatic elements may conform the control system 40 comprising at least two valves 42a, 42b, such that each valve 42a, 42b associated to a housing nozzle 22a, 22b is configured to control the flow of the gas 300 through each housing nozzle 22a, 22b.

[0087] The term “valve” refers to any mechanical or electromechanical device that regulates, directs, or controls the flow of a fluid (gases, liquids, fluidized solids, or slurries) by opening, closing, or partially obstructing one or more passageways.

[0088] The gas 300 may be connected from the gas inlet 30 to each valve 42a, 42b through one or more conduits 304a, 304b such as tubes.

[0089] Each valve 42a, 42b is associated to a housing nozzle 22a, 22b in the sense that each valve is configured to control the flow of the gas 300 through its associated housing nozzle 22a, 22b.

[0090] Each valve 42a, 42b is further configured to control the provision of the one or more components of the bioink 200 from each syringe 10a, 10b through its spray head nozzle 125a, 125b, by controlling the flow of gas 300 through its housing nozzle 22a, 22b. Each valve 42a, 42b may be connected to its housing nozzle 22a, 22b through one or more conduits 402a, 402b such as tubes. A shown with respect to Fig. 2 since each housing nozzle 22a, 22b is associated to a spray head 12a, 12b, and each spray head 12a, 12b comprises a spray head nozzle 125a, 125b, each valve 42a, 42b is associated to a spray head nozzle 125a, 125b. Through the flowing of gas 300 through its housing nozzle 22a, 22b, the provision of the one or more components of the bioink 200 from each syringe 10a, 10b is controlled through a venturi effect on the spray head nozzle 125a, 125b which generates the spray of the one or more components of the bioink 200 as droplets propelled by the gas 300.

[0091] As shown in Figs. 1 and 2, each spray head nozzle 125a, 125b is completely contained within its housing nozzle 22a, 22b.

[0092] It is noted that venturi effect is described as the reduction in fluid pressure that results when a moving fluid speeds up as it flows from one section of a pipe to a smaller section. Hence, when gas flows through the housing nozzle, there is a venturi effect within the housing nozzle 22a, 22b as its diameter is reduced, leading to a reduction in gas pressure along the housing nozzle 22a, 22b. Since each spray head nozzle 125a, 125b is completely contained within its housing nozzle 22a, 22b, the bioink 200 within each spray head nozzle 125a, 125b experiences this reduction in gas pressure, which creates a vacuum that generates the spray of the one or more components of the bioink 200 as droplets propelled by the gas 300.

[0093] It is noted that for simplicity purposes, Fig. 2 shows only part of the system 100. Thus, Fig. 2 does not disclose a control system 40 nor at least two nozzle heads 12a, 12b, nor at least two housing nozzles 22a, 22b, although the system 100 comprises such elements.

[0094] In a particular preferred embodiment, as shown with respect to Fig. 4, the system 100, further comprises the gas 300 and / or a gas source. The gas source may be a gas container, or an air pump. The person skilled in the art may envisage many different ways in which a gas source may be envisaged.

[0095] In a preferred embodiment of the first aspect of the invention, as shown with respect to Fig. 1 , the at least two valves 42a, 42b are configured to provide the one or more components of the bioink 200 of their respective syringe 10a, 10b independently.

[0096] Since each valve 42a, 42b controls the provision of the one or more components of the bioink 200 from the syringe 10a, 10b configured to be connected to the spray head 12a, 12b, associated to the housing nozzle 22a, 22b, in turn associated to the valve 42a, 42b, the one or more components of the bioink 200 from any of the at least two syringes 10a, 10b can be provided independently. Advantageously, this system provides a spray solution that enables its use in clinical practice, by enabling a simpler and easier to use way of providing different components of the bioink at the will of the clinician according to the necessities of the tissue receiving the bioink spray. For example, in some embodiments, a particular bioink may require the combination of a particular bioink with a substance that activates the bioink. Thus, the clinician may first provide the bioink in its inactive form as the first component of the bioink, and then provide the activation substance as the second component in a second stage, to activate the bioink once it has been provided.

[0097] In a more preferred embodiments, the at least two valves 42a, 42b are configured to provide the one or more components of the bioink 200 of their respective syringe 10a, 10b sequentially and / or simultaneously.

[0098] This further provides additional control on the provision of the one or more components of the bioink 200 which can further enable a simpler and easier to use way of providing the bioink 200 according to complex methodologies requiring sequential and / or simultaneous provision of one or more components of the bioink 200.

[0099] It is noted that Fig. 1 comprises many other elements, that are not comprised within the above-mentioned embodiments, and may be comprised in some preferred embodiments of the system 100, as will be described later on. For example, Fig. 1 shows references 32, 35, 44a, 44b. It is also noted that Figs. 1 and 2 are just schematical and the dimensions and shapes may differ in alternative embodiments of the above-mentioned embodiments of the first aspect of the invention. Moreover, Fig. 1 shows only two syringes 10a, 10b, but in other embodiments of the present invention the system may comprise more than two syringes. The same can be applied, mutatis, mutandis to the two spray heads 12a, 12b, and the two housing nozzles 22a, 22b.

[0100] In another preferred embodiment of the first aspect of the invention, and as shown with respect to Fig. 1 , one or more of the at least two valves 42a, 42b is an electric valve and controlled through an actuator 44a, 44b on the housing 20 surface.

[0101] An electric valve may be understood as a valve that is controlled by an electric current. Its operation can be influenced by electronic management systems or might be based on electromechanical principles. The electric valve encompasses a wide range of valve types, from those purely driven by electric motors to those that integrate electronic circuits for more refined control, and even those utilizing electromechanical processes, where electrical power is transformed into mechanical actions to adjust the valve's position. The actuator 44a, 44b may be any element actuatable by the user to control the one or more electric valves, such as buttons, pushbuttons, triggers, switches or the same. The skilled person may envisage many other alternatives for the actuator 44a, 44b at the light of this description, all of which are comprised within this disclosure. Each actuator may control one or more of the electric valves. For example, one actuator may control several valves simultaneously, while other actuator may control the operation of one single electric valve. Many different configurations may be foreseen at the light of this description, including actuators configured to actuate a plurality of valves in a predetermined manner. The actuator 44a, 44b may be electrically or mechanically connected to an electric valve 42a, 42b through a wire or mechanism 442a, 442b, such as a set of control wires, a trigger, a lever or any other mechanism the skilled person may envisage from the above disclosure.

[0102] Advantageously, the provision of one or more electric valves 42a, 42b controlled through one or more actuators 44a, 44b, simplifies the user interface and operational aspects of the system. Users can effortlessly decide which valve to actuate, ensuring a seamless and user-friendly experience. This approach provides enhanced precision and responsiveness in controlling the flow of gases through the valves, leading to more efficient and effective system operations.

[0103] In a more preferred embodiment, all of the at least two valves 42a, 44b, are electric valves.

[0104] In another preferred embodiment of the first aspect of the invention, and as shown for example in Fig. 3, at least one of the at least two spray head nozzles 125a, 125b comprises a diameter of 200pm and its associated housing nozzle 22a, 22b comprises a diameter of 500 pm.

[0105] Advantageously, as shown in Example 1 , the provision of a spray head nozzle 125a, 125b with a diameter of 200pm and its associated housing nozzle 22a, 22b a diameter of 500 pm, provides a desired configuration for the provision of a bioink associated to tissue regeneration.

[0106] It is noted that according to other embodiments of this preferred embodiment, the at least one of the at least two spray head nozzles 125a, 125b comprises a diameter of 200pm ± 15%, and its associated housing nozzle 22a, 22b comprises a diameter of 500 pm ± 15%. More preferably, the at least one of the at least two spray head nozzles 125a, 125b comprises a diameter of 200pm ± 10%, and its associated housing nozzle 22a, 22b comprises a diameter of 500 pm ± 10%. Even further preferably, at least one of the at least two spray head nozzles 125a, 125b comprises a diameter of 200pm ± 5%, and its associated housing nozzle 22a, 22b comprises a diameter of 500 pm ± 5%.

[0107] Advantageously, the provision of a spray head nozzle 125a, 125b with a diameter of 200pm ± 15%, preferably ± 10%, more preferably ± 5%; and its associated housing nozzle 22a, 22b a diameter of 500 pm ± 15%, preferably ± 10%, more preferably ± 5%, provides a desired configuration for the provision of a bioink associated to tissue regeneration.

[0108] In another preferred embodiment of the first aspect of the invention, the at least two syringes 10a, 10b are disposable syringes.

[0109] In another preferred embodiment of the first aspect of the invention, the system 100 further comprises the at least two syringes 10a, 10b.

[0110] The disposable syringes are single-use syringes designed to be used once and then discarded. They are typically made from plastic materials and come in various sizes and capacities to accommodate different applications.

[0111] Advantageously, this enables a bigger versatility, so that the system is readily adaptable when there is a need to change the components of the bioink. Also, since disposable syringes can come pre-filled, which eliminates the need for the user to handle bioink directly, reducing the potential for error and streamlining the process. Moreover, disposable syringes guarantee a sterile environment, minimizing the risk of contamination and ensuring compliance with stringent medical safety standards.

[0112] In another preferred embodiment of the first aspect of the invention, the housing 20 comprises Acrylonitrile Butadiene Styrene (ABS). Advantageously, the housing can be easily manufactured through 3D-printing techniques which in turn facilitates the creation of ergonomic and ad-hoc shape products that encompass the present invention.

[0113] In another preferred embodiment of the first aspect of the invention, and as shown with respect to Figs. 1 and 4, the system 100 further comprises a manometer 35 to regulate the pressure the gas 300 is provided through the gas inlet 30 to the system 100. The manometer may be connected to the gas 300 pipeline connected to the gas input 30 through a valve or regulator 353, or may be directly in series between the gas 300 and the gas input 32. While Fig. 1 shows the manometer 35 connected between a filter 32 and the gas input 30, the manometer 35 may be connected before the filter 32.

[0114] Advantageously, by adjusting and monitoring the pressure of the gas 300 entering through the gas inlet 30, it ensures that the bioink is delivered under optimal conditions, maintaining its integrity and efficacy of the bioink components. As shown in Example 1 it has been shown that controlling the gas 300 provision pressure is essential for some processes where bioinks are required.

[0115] In a more preferred embodiment of the first aspect of the invention, the gas 300 is provided with a pressure between 10 and 20 psi. As shown in Example 1 , a gas 300 with a pressure between 10 and 20 psi is needed for an appropriate bioink spray formation. A pressure higher than 20psi, leads to less cell viability, while a pressure lower than 10psi is insufficient to maintain a constant and steady flow of the sprayed material.

[0116] In a further preferred embodiment, the gas 300 is provided with a pressure between 12 and 18 psi, more preferably at 15 psi.

[0117] In another preferred embodiment of the first aspect of the invention, the system 100 further comprises a filter 32 between the gas inlet 30 and the gas 300.

[0118] Advantageously this ensures the sterility of the gas provided. Gases such as air, can develop microorganism that, if mixed with the bionic, can transform the spray into an infection vector. Since the bioink may be used with therapeutic uses, such as substituting or treating an affected surface, it is desirable to ensure that the gas driving the bioink over the surface is sterile. The filter 32 effectively serves as an additional safety layer, which can further configure the system 100 for medical applications.

[0119] In a more preferred embodiment of the first aspect of the invention, the filter 32 is a membrane filter. Even more preferably, the filter is a membrane filter of at most 0.25mm, even more preferred at most 0.22mm. The size of the membrane indicates the smallest size of particles that the membrane filter is capable of filtering. Advantageously, a membrane filter of at most 0.22mm ensures the housing 20 receives a sterile gas 300 through the gas inlet 30.

[0120] In another preferred embodiment of the first aspect of the invention, the viscosity of the bioink (200) is comprised within 0.5 mPa-s and 104mPa s.

[0121] Advantageously, a bioink 200 with a viscosity comprised between 0.5 mPa s and 104mPa s is optimally sprayed using a device according to one or more embodiments of the first aspect of the invention.

[0122] In a more preferred embodiment, the bioink 200 comprises Fib and / or FibD, the viscosity of the bioink (200) is comprised between 1.163 ± 0.095 mPa s for the Fib bioink; and / or from 1 ,114.75 mPa-s at shear rate y=0.1 s’1, to 2.97 mPa-s at shear rate y=800 s-1for the FibD bioink; at 25°C.

[0123] The symbol “y ” is the shear rate of each material. Fib has a Newtonian fluid behaviour, so the viscosity is kept constant (1 .163 ± 0.095 mPa s) at different shear rates. However, FibD has a shear-thinning fluid behaviour, so its viscosity decreases with higher shear-rates, thus the viscosity of FibD is presented as a range, depending on the shear rate.

[0124] In another preferred embodiment of the first aspect of the invention, the bioink 200 is a bioink formulation comprising fibrinogen and a glycosaminoglycans (GAGs) / collagen (Col) matrix, wherein the GAGs / Col matrix comprises at least a) 60 - 75% (w / w) hyaluronic acid, and b) 5 - 20% (w / w) sulphated GAGs, and c) 1 - 5% (w / w) of collagen.

[0125] Advantageously, a bioink with a formulation comprising fibrinogen, a GAGs / Col matrix and hMSCs and / or hDFs, wherein the GAGs matrix comprises at least 60 to 75% (w / w) hyaluronic acid, 5 to 20% (w / w) sulphated GAGs, and 1 to 5% (w / w) of collagen is the treatment of cutaneous wounds as described in Example 2-.

[0126] To this end two fibrinogen inks were formulated, one based on fibrinogen alone (Fib), and the other based on fibrinogen supplemented with GAGs / col matrix (FibD) as further detailed in Example 2. The ink is a source of GAGs / Col containing a high concentration of HA (67%), as well as sulfated GAGs (12%, including dermatan sulfate and chondroitin sulfate) and collagen (Col) (10.4%). These two inks were loaded with human mesenchymal stem cells (hMSCs) and human dermal fibroblasts (hDFs). The physicochemical properties of the bioinks, as well as their biocompatibility in vitro, were analyzed and found highly suitable for their intended application for spray application to the skin. Finally, their good skin wound healing properties were confirmed in an in vivo excisional wound healing murine model. Whilst in the examples the bioink was prepared mixing the GAGs / col matrix and the fibrinogen, it is to be understood that the components of the GAGs / col matrix HA, sulfated GAGs and collagen can also be separately added into the bioink formulation resulting in an equally suitable bioink.

[0127] In another preferred embodiment of the first aspect of the invention, the bioink 200 is a bioink formulation comprising fibrinogen and a GAGs / Col matrix, wherein the GAGs / Col matrix comprises hyaluronic acid, sulphated GAGs and collagen, and wherein (i) the final amount of hyaluronic acid in the bioink formulation is between about 0.3 - 37.5 mg / ml or 0.03 - 3.75 % (w / v),

[0128] (ii) the final amount of sulphated GAGs in the bioink formulation is between about 0.025 - 10 mg / ml or 0.0025 - 1.0 % (w / v) sulphated GAGs, and

[0129] (iii) the final amount of collagen in the bioink formulation is between about 0.005 - 2.5 mg / ml or 0.0005 - 0.25 % (w / v).

[0130] In one embodiment of the GAGs / Col matrix comprises at least a) 60 - 75% (w / w) hyaluronic acid, and b) 5 - 20% (w / w) sulphated GAGs, and c) 1 - 5% (w / w) of collagen.

[0131] This GAGs / Col matrix corresponds to Dermial® (Bioiberica).

[0132] As detailed in Example 3 a), the Fib and FibD bioinks of present invention presented acidic pH values of 6.12 ± 0.19 and 6.10 ± 0.17, respectively, adequate for their topical application. The swelling or liquid absorption ability of hydrogels influences the entrance of fluids and the transport of nutrients into the hydrophilic hydrogel matrices. The Fib and FibD hydrogels presented a satisfactory swelling capacity after just two days (Example 3 b)). The FibD hydrogels showed a higher swelling ratio in comparison to the Fib hydrogels, expected due to the presence of high concentrations of HA in the GAGs / Col matrix component, given the high-water retention capability of HA.

[0133] On the other hand, the biodegradability of the hydrogels is important as it affects cell growth and tissue regeneration. As detailed in Example 3 c) the Fib hydrogels degraded faster than FibD hydrogels, probably owing to the more complex matrix formed in the FibD hydrogels as a result of the different biomolecules of GAGs / Col matrix. All Fib hydrogels had degraded completely after 17 weeks, whereas FibD hydrogels degraded after 20 weeks (i.e. Fib hydrogels degraded 15% faster than the FibD hydrogels).

[0134] In biofabrication, a high viscosity of the bioink or biomaterial ink is essential to maintain the 3D structure of the resulting construct, and specifically in skin sprays, to prevent run off of the sprayed material and prolong its contact time with the wound bed; but as mentioned previously, a higher viscosity of the transporting fluid affects cell viability negatively. Fibrinogen inks have been reported to exhibit a Newtonian fluid behavior, which is congruous with the results, where the Fib ink maintained a constant viscosity of 1.163 ± 0.095 mPa-s (Example 3 d)). On the other hand, the supplementation of the Fib ink with GAGs / Col matrix resulted in a shear-thinning behavior of the FibD ink. This shear-thinning behavior is favorable for the use of bioinks in biofabrication techniques such as skin sprays or 3D bioprinting, given that the decrease in viscosity under high shear stress (during extrusion) reduces cell damage, and the subsequent recovery of high viscosity under low shear stress (after extrusion) helps to prevent leakage of the material and maintain the 3D shape.

[0135] In a particular embodiment, the bioink 200 is a bioink formulation further comprises human cells, preferably human mesenchymal stem cells (hMSCs), keratinocytes and / or human dermal fibroblasts (hDFs).

[0136] In another particular embodiment, the bioink 200 is a bioink formulation of present invention wherein

[0137] (i) the final amount of hyaluronic acid in the bioink is between about 0.6 - 18.75 mg / ml or 0.06 - 1 .875 % (w / v),

[0138] (ii) the final amount of sulphated GAGs in the bioink is between about 0.05 - 5 mg / ml or 0.005 - 0.5 % (w / v) sulphated GAGs, and

[0139] (iii) the final amount of collagen in the bioink is between about 0.01 - 1.25 mg / ml or 0.001 - 0.125 % (w / v).

[0140] The final amount of hyaluronic acid in the bioink can be between about 0.4 - 35 mg / ml or 0.04 - 3.5 % (w / v), between about 0.5 - 25 mg / ml or 0.05 - 2.5 % (w / v) or between about 0.6 - 20 mg / ml or 0.06 - 2.0 % (w / v).

[0141] The final amount of sulphated GAGs in the bioink formulation can be between about 0.03 - 8 mg / ml or 0.004 - 0.8 % (w / v) sulphated GAGs, between about 0.04 - 7 mg / ml or 0.004 - 0.7 % (w / v) sulphated GAGs, or between about 0.04 - 6 mg / ml or 0.004 - 0.6 % (w / v) sulphated GAGs

[0142] The final amount of collagen in the bioink can be between about 0.007 - 2.0 mg / ml or 0.0007 - 0.20 % (w / v), between about 0.008 - 1 .5 mg / ml or 0.0008 - 0.15 % (w / v), or between about 0.009 - 1.4 mg / ml or 0.0009 - 0.14 % (w / v). It is to be understood that the bioink of present invention is not prepared from patients cells but is prepared by mixing the herein specified components together as also described in the examples section.

[0143] The benefit of this bioink as opposed to bioinks of the prior art that are prepared using a decellularized extracellular matrix (dECM) is that its preparation is very simple, does not require the preparation via cells and is suitable for use in any patient.

[0144] Unlike dECM-based bioinks, the bioink of the present invention does not contain undefined ECM components derived from decellularization processes, ensuring greater reproducibility and regulatory compliance.

[0145] Specifically, it excludes:

[0146] • Endogenous residual growth factors and cytokines: dECM retains bioactive molecules from its source tissue, which can introduce batch-to-batch variability and affect cell behaviour in unpredictable ways. Our bioink relies on a defined composition, ensuring controlled and reproducible cellular responses.

[0147] • Cell membrane fragments and nuclear residues: Even with optimized decellularization, traces of cellular components may persist in dECM, potentially triggering immune responses or requiring additional regulatory scrutiny. Our bioink is free from such contaminants.

[0148] • Tissue-specific ECM components of unknown composition: dECM varies depending on its source (e.g., donor age, anatomical site), which can lead to inconsistencies in mechanical and biochemical properties. Our bioink contains a standardized GAG / Col matrix, ensuring consistent performance across batches.

[0149] • Residual decellularization reagents: Chemical or enzymatic agents used for decellularization, such as detergents (e.g., SDS, Triton X-100) or enzymes (e.g., trypsin, DNase), may leave behind traces that could impact cell viability or require additional purification steps. Our bioink, being composed of well-defined components, eliminates this concern.

[0150] By excluding these dECM-specific components, the bioink of present invention offers a more controlled, reproducible, and regulatory-friendly alternative. In one embodiment the bioink of present invention only consist of fibrinogen and the GAGs / Col matrix. In preferred embodiment the bioink of present invention consist of fibrinogen, the GAGs / Col matrix and human cells, preferably human mesenchymal stem cells (hMSCs), keratinocytes and / or human dermal fibroblasts (hDFs).

[0151] In one preferred embodiment the bioink of the present invention does not comprise a decellularized extracellular matrix (dECM).

[0152] In another preferred embodiment, the bioink 200 of the present invention does not comprise any one of endogenous residual growth factors, cytokines, cell membrane fragments, nuclear residues, tissue-specific ECM components, residual decellularization reagents, or any combination thereof.

[0153] In a more preferred embodiment of the first aspect of the invention, the amount of fibrinogen in the formulation is between 1 and 100 mg / ml. Additionally or alternatively, the amount of fibrinogen in the formulation is between 0.1 and 10 % (w / v). This amount of fibrinogen provides adequate viscosity and mechanical properties for the application, preferably as a spray, of the bioink onto the surface. Furthermore, the amount of fibrinogen allows, once mixed with thrombin, the formation of hydrogels in which the fibrin fibers form a matrix with a good density and porosity to allow the correct attachment, proliferation and viability of the cells.

[0154] In one embodiment the amount of fibrinogen in the formulation is between 2 and 90 mg / ml, between 3 and 80 mg / ml, between 4 and 70 mg / ml, between 5 and 60 mg / ml, between 6 and 50 mg / ml, between 7 and 40 mg / ml, between 8 and 30 mg / ml, between 9 and 20 mg / ml. In a preferred embodiment of the bioink formulation of present invention the amount of fibrinogen is about 10 mg / ml.

[0155] In one embodiment of the bioink formulation of present invention the amount of fibrinogen in the formulation is between 0.1 and 10% (w / v), between 0.2 and 9% (w / v), between 0.3 and 8% (w / v), between 0.4 and 7% (w / v), between 0.5 and 6% (w / v), between 0.6 and 5% (w / v), between 0.7 and 4% (w / v), between 0.8 and 3% (w / v), between 0.9 and 2% (w / v). In a preferred embodiment of the bioink formulation of present invention the amount of fibrinogen is about 1% (w / v).

[0156] In another more preferred embodiment of the first aspect of the invention, the bioink formulation of present invention the amount of GAGs / Col matrix in the formulation is between 1 and 25 mg / ml. Additionally or alternatively, the amount of GAGs / Col matrix in the formulation is between 0.1 and 2.5% (w / v).

[0157] This amount of GAGs / Col matrix provides for higher viability and number of cells. Furthermore, the GAGs / Col matrix provides better rheological properties to the bioink, as in terms of viscosity, the fibrinogen only bioink (Fib) presents a Newtonian behaviour, while the fibrinogen / GAGs / Col matrix (FibD) bioink shows a shear-thinning behaviour.

[0158] In one embodiment of the bioink formulation of present invention the amount of GAGs / Col matrix in the formulation is between 1.1 and 20 mg / ml, between 1.2 and 15 mg / ml, between 1.3 and 10 mg / ml, between 1.4 and 5 mg / ml, between 1.5 and 4 mg / ml, between 1.6 and 3 mg / ml.

[0159] In a preferred embodiment the amount of GAGs / Col matrix in the formulation is 1.7 mg / ml. In an even more preferred embodiment, the amount of GAGs / Col matrix in the formulation is 2.5 mg / ml.

[0160] In another more preferred embodiment of the first aspect of the invention, the amount of hyaluronic acid in the GAGs / Col matrix is between 65 and 70% (w / w), between 66 and 69% (w / w), between 67 and 68% (w / w). In a preferred embodiment the amount of hyaluronic acid in the GAGs / Col matrix is about 67% (w / w).

[0161] In another more preferred embodiment of the first aspect of the invention, the amount of collagen in the GAGs / Col matrix is between 5 and 15% (w / w), preferably between 8 and 12% (w / w). In a preferred embodiment the amount of collagen in the GAGs / Col matrix is between 9 and 11 % (w / w), most preferred about 10% (w / w).

[0162] In another more preferred embodiment of the first aspect of the invention, the sulphated GAGs in the GAGs / Col matrix are selected from dermatan sulphate and / or chondroitin sulphate.

[0163] In another more preferred embodiment of the first aspect of the invention, the amount of sulphated GAGs in the GAGs / Col matrix is between 10 and 15% (w / w), between 11 and 14% (w / w), preferably between 11 and 13% (w / w), most preferred about 12% (w / w).

[0164] In another more preferred embodiment of the first aspect of the invention, the bioink formulation of present invention further comprises other components that accommodate different functionalities. In one embodiment the bioink formulation comprises masclinic acid nanoparticles, preferably solid lipid nanoparticles (SLNs). Maslinic acid (MA) is a pentacyclic triterpene that exhibits antitumor, antioxidant, anti-inflammatory, antidiabetic, antiparasitic, cardioprotective and neuroprotective properties and can therefore be used in the bioink of present invention to provide these additional functionalities to the bioink. In one embodiment the MA nanoparticles comprise a shell, preferably a shell comprised of Poloxamer 407 (PMA), dicarboxylic acid-Poloxamer 407 (PCMA), HA-coated PCMA (PCMA-HA), or combinations thereof.

[0165] In another preferred embodiment of the first aspect of the invention as shown by Fig. 8, the control system 40 further comprises the features according to the second aspect of the invention.

[0166] Advantageously, this allows for a system for providing a bioink in the form of a spray and by extrusion, which further improves the versatility of the device to provide the bioink as required either in the form of spray or as an extruded construct.

[0167] In another preferred embodiment of the first aspect of the invention, the system 100 is configured to provide a bioink 200 comprising a shear rate comprised between 0.1 and 15.000 s-1and a viscosity comprised between 1 and 300.000 mPa s at a shear rate of 1000 s-1; at a pressure comprised between 13.8 and 241.3 kPa. As shown in Example 4, shear stress, pressure, and viscosity are key parameters that are interconnected and have a critical role in maintaining the survival and the proper metabolism of the cells during the printing process, and a bioink 200 within these parameters ranges ensure that bioinks can be effectively provided using a system according to the first aspect of the invention.

[0168] In another more preferred embodiment when the system 100 is for providing a bioink 200 in the form of a spray, the system 100 is configured to provide a bioink 200 comprising a shear rate comprised between 1 and 15,000 s-1and a viscosity comprised between 1 and 300,000 mPa s at a shear rate of 1 ,000 s-1; at a pressure comprised between 13.8 and 241.3 kPa. As shown in Example 4, a bioink 200 within these parameters ranges ensure that bioinks can be effectively provided in the form of a spray using a system according to the first aspect of the invention.

[0169] In a more preferred embodiment, when the system 100 is for providing a bioink 200 by extrusion, the system 100 is configured to provide a bioink 200 comprising a shear rate comprised between 0.1 and 500 s-1and a viscosity comprised between 1 and 300,000 1 mPa-s at a shear rate of 1 ,000 s-1; at a pressure comprised between 13.8 and 241.3 kPa. As shown in Example 4, a bioink 200 within these parameters ranges ensure that bioinks can be effectively provided by extrusion using a system according to the first aspect of the invention.

[0170] A second aspect of the invention relates to a system for providing a bioink by extrusion. As shown with reference to Fig. 8 the system 100 comprises at least two spray heads 12a, 12b and a housing 20 according to the first aspect of the invention. All the definitions and descriptions of the first aspect of the invention therefore apply mutatis mutandis to the second aspect of the invention.

[0171] The system 100 of the second aspect of the invention further comprises a control system 40 comprising at least two actuators 43a, 43b each actuator 43a, 43b associated to a housing nozzle 22a, 22b.

[0172] Each actuator 43a, 43b is configured to control the provision of the one or more components of the bioink 200 from each syringe 10a, 10b through its housing nozzle 22a, 22b by extrusion.

[0173] The control system 40, although represented as a rectangle in Fig. 8, is a sophisticated assembly designed to manage the system operation. It can integrate a myriad of components, spanning across the electronic, electric, and pneumatic domains. The skilled person may envisage many ways in which electronic, electric, and / or pneumatic elements may conform the control system 40 comprising at least two valves 42a, 42b, such that each actuator 43a, 43b is configured to control the provision of the one or more components of the bioink 200 from each syringe 10a, 10b through its housing nozzle 22a, 22b. For example, the actuator 43a, 43b may be a mechanical actuator configured to slide the plunger of the syringe in order to control the provision of the one or more components of the bioink 200 from each syringe 10a, 10b.

[0174] It is noted that Fig. 8 comprises many other elements, that are not comprised within the above-mentioned embodiments, and may be comprised in some preferred embodiments of the system 100, as will be described later on. For example, Fig. 1 shows references 32, 35, 44a, 44b, 50, 60. It is also noted that Fig. 8 is just schematical and the dimensions and shapes may differ in alternative embodiments of the above-mentioned embodiments of the first aspect of the invention. Moreover, Fig. 8 shows only two syringes 10a, 10b, but in other embodiments of the present invention the system may comprise more than two syringes. The same can be applied, mutatis, mutandis to the two spray heads 12a, 12b, and the two housing nozzles 22a, 22b.

[0175] In a preferred embodiment of the second aspect of the invention, as shown with respect to Fig. 8, the at least two actuators 43a, 43b are configured to provide the one or more components of the bioink 200 of their respective syringe 10a, 10b independently.

[0176] It is noted that all of the preferred embodiments of the first aspect of the invention also apply, mutatis mutandis to the second aspect of the invention.

[0177] Hence, according to the first and second aspects of the inventions, the present invention refers to a system 100 for providing a bioink 200, comprising: a) at least two spray heads 12a, 12b, each a spray head 12a, 12b configured to receive a syringe 10a, 10b, each syringe 10a, 10b configured to receive one or more components of the bioink 200 and wherein each a spray head 12a, 12b comprises a nozzle 125a, 125b, b) a housing 20, configured to comprise the at least two spray heads 12a, 12b, and comprising at least two housing nozzles 22a, 22b, each housing nozzle 22a, 22b associated to a spray head 12a, 12b and disposed around the spray head nozzle 125a, 125b, the housing nozzle 22a, 22b comprising a diameter bigger than the diameter of the spray head nozzle 125a, 125b, c) a gas inlet 30 connected to the housing (20) configured to receive a gas 300, and a control system 40. The control system comprises at least one of the following two alternatives: i. when the system is for providing a bioink 200 in the form of a spray the control system, comprises at least two valves 42a, 42b, each valve 42a, 42b associated to a housing nozzle 22a, 22b and configured to control the flow of the gas 300 through each housing nozzle 22a, 22b, wherein each valve 42a, 42b is further configured to control the provision of the one or more components of the bioink 200 from each syringe 10a, 10b through its spray head nozzle 125a, 125b, by controlling the flow of gas 300 through its housing nozzle 22a, 22b, and wherein the at least two valves 42a, 42b are configured to provide the one or more components of the bioink 200 of their respective syringe 10a, 10b independently; or ii. when the system is for providing a bioink 200 by extrusion, the control system comprises at least two actuators 43a, 43b each actuator 43a, 43b associated to a housing nozzle 22a, 22b, wherein each actuator 43a, 43b is configured to control the provision of the one or more components of the bioink 200 from each syringe 10a, 10b through its housing nozzle 22a, 22b by extrusion; and wherein the at least two actuators 43a, 43b are configured to provide the one or more components of the bioink 200 of their respective syringe 10a, 10b independently.

[0178] Advantageously, this allows for a system for providing a bioink in the form of a spray and / or by extrusion, which further improves the versatility of the device to provide the bioink as required either in the form of spray or as an extruded construct.

[0179] In another preferred embodiment of the second aspect of the invention, as shown with respect to Fig. 8, the system 100, further comprises an UV light 50 for photocuring the bioink 200. Bioinks 200 can be photocured in the presence of a photoinitiatior such as gelatine methacrylate (GelMA), collagen methacrylate (ColMA), Hyaluronic Acid Methacrylate (HAMA), Silk Methacrylate (SilMA), or Polyethylene Glycol Diacrylate (PEGDA). The UV light may be controlled by a controller to be lighted after an instruction form the user, for example after certain time after the bioink has been provided or if a dedicated button is pressed.

[0180] In another preferred embodiment of the second aspect of the invention, as shown with respect to Fig. 8, the system 100, further comprises a camera 60, more preferably a high resolution camera, even more preferably a 4K camera. Advantageously, this improves the visualization of the field to be treated.

[0181] A third aspect of the invention relates to a method of proving a bioink 200 for the treatment of tissue injuries or damages.

[0182] As shown in Fig. 5, the method comprises a first step 501 of providing at least two syringes 10a, 10b, each syringe 10a, 10b comprising one or more components of the bioink 200 in a system 100 according to any one of the embodiments of the first aspect of the invention.

[0183] A second step 502 involves actuating one or more of the at least two valves 42a, 42b sequentially and / or simultaneously to provide the bioink 200 in the form of a spray over the tissue to be treated. Advantageously, the proposed method of providing a bioink 200 for the treatment of tissue injuries or damages allows for a method with higher control on the provision of the bioink, such that a particular bioink provision method can be followed, wherein each of the one or more components of the bioink can be provided sequentially and / or simultaneously. Therefore, one of the distinguishing advantages is the method's ability to independently supply individual bioink components. This flexibility ensures that the treatment can be precisely tailored to meet specific requirements.

[0184] Alternatively or additionally, the second step 502 involves actuating one or more of the at least two actuators (43a, 43b) sequentially and / or simultaneously to provide the bioink (200) by extrusion over the tissue to be treated.

[0185] For instance, consider a scenario in wound healing. The tissue might first require a layer from syringe 10a, which contains one or more bioink components that initiates cellular adhesion and establishes a primary scaffold. Once this layer is settled, a second set of one or more components from syringe 10b might then be sprayed or extruded to provide growth factors and stimulate cellular proliferation, accelerating the healing process. By controlling the sequence of component delivery, the method ensures optimal interaction of the components on the tissue, potentially optimizing therapeutic outcomes.

[0186] Also, there might be instances where a tissue needs immediate exposure to multiple bioink components to achieve the desired therapeutic outcome. In such cases, the method is equipped to deliver components from both syringes 10a and 10b simultaneously. An example might be a scenario where a tissue requires both structural support and immediate nutrient supply. The method's capacity to concurrently spray components ensures the tissue receives the combined benefits of both sets of one or more components of the bioink form the at least two syringes 10a, 10b without delay.

[0187] The method according to the third aspect of the invention holds promise for addressing various tissue injuries or damages. A prime example is skin damage. Whether it's due to burns, ulcers, abrasions, or surgical procedures, the skin often requires prompt and tailored treatment. Using the method described, clinicians can deliver specific bioink components, either sequentially or simultaneously, directly to the affected area. This ensures that the tissue receives exactly what it needs at the precise moment it's needed to effectively treat the tissue injury or damage. As previously discussed, the ability to control which bioink components are delivered, and in what order, allows for highly customized treatments. This is particularly crucial when treating complex wounds that might require a staged approach for optimal healing. It is noted, that when the method is associated to a bioink formulation comprising fibrinogen, a glycosaminoglycans (GAGs) / Collagen (Col) matrix and optionally with human mesenchymal stem cells (hMSCs) and / or human dermal fibroblasts (hDFs), wherein the GAGs / Col matrix comprises at least 60 to 75% (w / w) hyaluronic acid, 5 to 20% (w / w) sulphated GAGs, and 1 to 5% (w / w) of collagen, the method is significantly suitable for treatment of the skin. As shown in Example 3, the physicochemical properties of the bioinks, as well as their biocompatibility in vitro, were analyzed and found highly suitable for their intended application for spray application to the skin. Finally, their good skin wound healing properties were confirmed in an in vivo excisional wound healing murine model.

[0188] All of the above are fully within the scope of the present disclosure, and are considered to form the basis for alternative embodiments in which one or more combinations of the abovedescribed features are applied, without limitation to the specific combination disclosed above.

[0189] In light of this, there will be many alternatives which implement the teaching of the present disclosure. It is expected that one skilled in the art will be able to modify and adapt the above disclosure to suit its own circumstances and requirements within the scope of the present disclosure, while retaining some or all technical effects of the same, either disclosed or derivable from the above, in light of his common general knowledge in this art. All such equivalents, modifications or adaptations fall within the scope of the present disclosure.

[0190] EXAMPLES

[0191] Example 1 : Spray device

[0192] A novel airbrush-based spraying device was designed to support dual extrusion, controlled by a pneumatic system (Figure 4). Placed on each side of the device, two on / off buttons (one for each material) connected to electronic valves open / close the airflow from the air pump (Figure 4). By pressing one of the On / Off buttons or both at the same time, both solutions can be sprayed either subsequently or simultaneously, respectively. A pressure regulator placed at the air pump controls air pressure, and consequently, the material deposition rate. By increasing the air pressure, more force is exerted, and a larger volume of air pushes the material out through the nozzle. The material is extruded through an inner 200 pm-diameter nozzle, while air is pushed through an outer 500 pm-diameter nozzle (Figure 3). When the material meets the air stream near the outlet orifice of the nozzles, the material is pulverized generating bioink droplets (Figure 2).

[0193] An AlamarBlue HS® assay was used to determine the best spraying pressure (Figure 6). Metabolic activity of hDFs decreased with increasing spraying pressure, evidencing how cells are damaged with increasing pressures. Although the lowest pressure (10 psi) showed the highest metabolic levels, this pressure proved to be insufficient to maintain a constant and steady flow of the sprayed material. Therefore, a spraying pressure of 15 psi was chosen for the following experiments as it still showed high metabolic activity levels, close to those of 10 psi.

[0194] Discussion

[0195] In this study, we have developed a novel spray system for the treatment of cutaneous wounds. To date, the vast majority of skin spray studies utilize syringes with spray pumps or regular airbrushes, which can only spray one material at a time; and the few spraying devices that allow the delivery of two materials (generally fibrinogen and thrombin solutions) are double syringes attached to spray tips. We have developed the first dual-extrusion airbrush-based device designed for the treatment of skin lesions, with a double spraying system that allows the delivery of two different materials either simultaneously or subsequently. Furthermore, the syringes can be quickly and easily replaced for others loaded with diverse bioinks, which could be useful in case of wanting to spray subsequent layers with different composition, in order to simulate the different layers of the skin. Given the numerous advantages for wound healing, our bioinks were formulated with fibrinogen, which has been the most widely used biomaterial for skin sprays due to its hemostatic properties, and supplemented with GAGs / Col matrix, which provides a source of GAGs, mainly HA but also dermatan sulfate, and collagen. In addition to the benefits of fibrinogen, HA promotes tissue regeneration during the wound healing process, reduces scar formation, maintains skin moisture, and has immunomodulatory properties. Moreover, GAGs / Col matrix has been reported to possess regenerative, anti-aging and antioxidant properties in vitro, and stimulate the synthesis of collagen, elastin, and GAGs. This is the first reported bioink based on a fibrinogen-HA combination for application in skin sprays.

[0196] A frequent drawback of many skin spray studies is the little importance they give to the spraying parameters, as the use of different parameters can influence results significantly, especially in cell sprays. Depending on the duration, cells are generally damaged by hydrostatic, shear, and elongation stresses while being sprayed and passing through the nozzle, but they are also damaged when they impact on the receiving surface. Cell viability has been reported to be negatively affected with a smaller nozzle diameter, higher spraying pressure and velocity, higher viscosity of the transporting fluid, and stiffness of the receiving surface. On the contrary, a larger cell-containing droplet diameter and a longer spraying distance are favorable for cell viability. Although the viability of epidermal cells after spraying has been demonstrated in several studies, cells can be damaged without having their membrane disrupted, so the post-aerosolization proliferative capacity must be assessed as well as the viability. Harkin et al. reported that the viability and mitochondrial enzyme activity of keratinocytes immediately after aerosolization was reduced with spraying pressures above 20 psi. We optimized the spraying pressure of our system to 15 psi, as higher pressures resulted in a decreased metabolic activity and proliferative capacity. For our experiments, we used an acetate cone as a funnel to keep better control of the sprayed volume. However, this would not be necessary in clinical use, as the area to be sprayed would generally be more extensive.

[0197] Example 2: Formulation of fibrinogen-based bioinks

[0198] Two different bioinks were formulated, one consisting of fibrinogen alone and another consisting of fibrinogen supplemented with GAGs / Col matrix. To prepare the fibrinogen (Fib) ink, bovine fibrinogen (Sigma-Aldrich) was diluted in 0.9% NaCI at 37°C, to obtain a final fibrinogen concentration of 10 mg / ml. Once dissolved, the solution was filtered through 0.22 pm membrane filters (Merck Millipore) to ensure sterility. For the fibrinogen- GAGs / Col matrix (FibD) ink, previously UV-sterilized GAGs / Col matrix was added to the Fib solution at a final concentration of 2.5 mg / mL. In case of preparing cell-loaded hydrogels, hMSCs or hDFs were then gently mixed with the Fib or FibD inks at a density of T 106cells / mL. As well, bovine thrombin (Sigma-Aldrich) was diluted in 40mM CaCh at a final concentration of 50 U / rnL. Finally, the fibrin-based hydrogels were obtained by mixing the Fib or FibD bioinks with the thrombin solution in a 10:1 (v / v) ratio, respectively, and letting them gel for at least 5 minutes at 37°C.

[0199] Example 3: Determination of Physicochemical properties a) pH determination

[0200] The pH of the Fib and FibD inks was measured (n=6) with a calibrated digital pH-meter Hach Sension+ (Hach Lange S.L., Spain) at room temperature. The pH values of the Fib and FibD inks were 6.12 ± 0.19 and 6.10 ± 0.17, respectively. b) Swelling test of Fib and FibD hydrogels Previously freeze-dried hydrogels were weighed, submerged in PBS, and kept at 37°C. At determined time points, samples (n=6) were taken out, absorbing excess of PBS with filter paper, and weighed. The swelling rates (%) were calculated as a measure of weight gain with the following equation:

[0201] Swelling ratio (%) = x 100 (1)

[0202] Wois the initial wet weight of samples (t = 0 h).

[0203] Wtis the wet weight of samples at the corresponding time point (t).

[0204] The swelling behavior of previously freeze-dried Fib and FibD hydrogels was observed by immersing the hydrogels in PBS and measuring weight increase (Figure 7A). For both Fib and FibD hydrogels, the swelling ratio increased during the first 48h, reaching an average swelling of 66.7 ± 10.2 % and 72.2 ± 5.0 % for Fib and FibD, respectively. Afterwards, hydrogels’ weight started decreasing slightly due to degradation. The swelling ratio of the FibD hydrogels was higher than Fib at all time points, but no significant differences were observed. c) Degradation test of Fib and FibD hydrogels

[0205] Pre-weighed hydrogels were immersed in PBS and maintained at 37°C under gentle agitation. At determined time intervals, samples (n=6) were centrifuged at 3000 G for 10 minutes, the supernatant was removed, and hydrogels were weighed. The degradation rates (%) as a measure of weight loss were calculated with the following equation: x 100 (2)

[0206] WQis the initial wet weight of samples (t = 0 h).

[0207] Wtis the wet weight of samples at the corresponding time point (t).

[0208] The degradation rate of hydrogels was analyzed by measuring weight loss over time (Figure 7B). The degradation of Fib and FibD hydrogels increased rapidly during the first week, and then kept increasing moderately every week. The Fib hydrogels degraded faster than the FibD hydrogels, presenting a higher degradation ratio during all the experiment. All Fib hydrogels had degraded completely after X days, whereas FibD hydrogels required X days to degrade. d) Mechanical and rheological characterization Rheological assays were carried out in order to obtain information on the viscosity of the fibrin-based inks, as well as on the compression and shearing characteristics of the hydrogels, using a torsional rheometer MCR302 (Anton Paar, Austria). All the rheological assays were performed in triplicate for all conditions and at a temperature of 25°C.

[0209] The shear viscosity was obtained using a cone-plate geometry (50 mm diameter and 1 ° angle). Samples of each material (distilled water, NaCI 0.9%, and the Fib and FibD inks) were sequentially placed on the base of the rheometer. First, they were rotatory pre-sheared at a constant shear rate of 800 s-1for 1 min. Then, they were allowed to rest for 1 min, with no shear rate applied. Lastly, they were rotatory sheared with a logarithmic shear rate ramp from 0.01 to 800 s-1(acquisition time of 5 seconds) for 5 min.

[0210] A plate-plate geometry (20 mm diameter and 5 mm gap) was used to analyze the compression and oscillatory shear behavior of hydrogels (prepared with a diameter of 20 mm and a height of approximately 5 mm) and mice skin samples (cut in a circular form with the same diameter, 20 mm). First, samples were placed onto the base of the rheometer and were approached by the rheometer head at a constant speed of 10 pm / s, up to a normal force of 0.1 N (in the case of the hydrogels) or 0.5 N (in the case of the mice skin), to determine the Young's modulus. Secondly, the normal force was kept constant for 30 seconds at 0.1 N or 0.5 N, correspondingly. Finally, to determine the shear viscoelastic moduli, the samples were oscillatory sheared according to a strain amplitude of 0.001 % to 1000% at a frequency of 1 Hz and a normal force of 0.1 N (for hydrogels) or 0.5 N (for mice skin).

[0211] In order to assess the capability of the inks to be sprayed through the airbrush's nozzle, the shear viscosity of the Fib and FibD inks, as well as distilled water and 0.9% NaCI (the basis of the fibrin inks formulation), was determined with a rotational shear test (Figure 7C). At 25°C, water and NaCI obtained viscosity results of 0.871 ± 0.039 and 0.896 ± 0.272 mPa s, respectively. As expected, they demonstrated a Newtonian flow behavior, with viscosity being independent of the shear rate. Interestingly, despite being a polymeric solution, the Fib ink also showed a Newtonian behavior, with a viscosity of 1.163 ± 0.095 mPa s. However, the addition of GAGs / Col matrix (which contains high concentrations of HA molecules) resulted in the FibD ink having a shear-thinning behavior, with a decreasing viscosity with increasing shear rates (ranging from 1 ,114.75 mPa s at y -0.1 s-1 , to 2.97 mPa s at y =800 s-1), typical of polymeric solutions.

[0212] On the other hand, the compression and oscillatory shearing characteristics of the hydrogels formed from the bioinks are analyzed in Figures 2D, E and F. Manually created (pipetted) Fib and FibD hydrogels (Control) were compared with sprayed Fib and FibD hydrogels, respectively, with no significant differences found. Between Fib and FibD hydrogels, differences were not statistically significant, but higher Storage (Figure 7E) and Loss (Figure 7F) moduli could be observed for the FibD hydrogels than the Fib ones, but this difference was not observable in cell-loaded hydrogels. There were significant differences in the Young's Modulus between sprayed acellular FibD hydrogels and sprayed hDFs-loaded FibD hydrogels, both after 1 and 21 days (Figure 7D); as well as in the Storage and Loss moduli between sprayed acellular Fib hydrogels and sprayed hDFs-loaded Fib hydrogels after 21 days in culture; and between sprayed acellular FibD hydrogels and hDFs- loaded FibD hydrogels after 21 days (Figures 2E and F). For hDFs and hMSCs-loaded hydrogels, both with Fib and FibD, differences were observed when comparing hydrogels maintained in culture for 1 or 21 days: hydrogels cultured for 21 days showed higher Young's moduli (Figure 7D) and lower Viscoelastic moduli (Figures 2E and F), than those cultured for 1 day. Finally, samples of healthy skin of mice revealed higher Young's and Viscoelastic moduli than all hydrogels, but the difference was not statistically significant for the Young's Modulus.

[0213] Example 4: Optimization of a Multifunctional Hand-held 3D Biopen System for use in Regenerative Medicine

[0214] 1. Introduction

[0215] Despite being the largest organ of humans’ body and being one of the main barriers responsible of protection, the skin is extremely vulnerable to damage. After mechanical injuries, burns, or even chronic skin trauma associated to diseases like diabetes, a complex and dynamic recovery process starts. In the case of deep partial-thickness or full-thickness injuries, the skin’s own regenerative components are destroyed, so it loses its regenerative potential [21-25], In order to avoid a late re-epithelization, this kind of wound needs to be immediately treated. Otherwise, the result involves impaired cell function and consequently, the failure of normal healing

[0026] , Many standard wound healing procedures are being daily implemented in hospitals. For example, the split-thickness autograft is frequently used for chronic wound treatment. It consists on harvesting a full-thickness fascia from a donor site and grafting it over the affected region

[0027] , The main limitation of this technique is the amount and the quality of donor skin that is available in the patient. Furthermore, the risk of suffering infections and other complications can increase significantly. Another re-markable approach is using cultured epithelial autografts. They are based on culturing autologous keratinocytes extracted from a biopsy of the patient, to finally graft the cultured epithelia onto the wound bed

[0028] , The success of the graft depends majorly on its proper attachment in the basement membrane, and the main drawback is the time required to culture and prepare sheets of cells for grafting [23,25,29], The limitations of the current treatments have boosted the research in the sense of finding biomaterials and techniques that contribute to full skin recovery; not only related to the correct structural stratification of the different layers, but also related to the proper regeneration of its original function [30,31],

[0216] One promising strategy for wound healing is using an artificial matrix as the wound dressing

[0032] , Through this methodology, it is possible to preserve the hydration of the compromised area and optimize the skin regeneration, to avoid infections and disruptions of the wound base

[0022] , Considering this, a variety of scaffold formats have been developed (films, foams, nanofibers...), but recently, hydrogels have gained much attention in the field of skin regeneration [33-35], These are 3D networks com-posed of hydrophilic polymers whose structure mimics the extracellular matrix (ECM), which have tunable mechanical properties and facile bioactive substance delivery capability [36,37],

[0217] Many polymeric materials have been evaluated for skin tissue engineering (TE) purposes, which has led to the development of multiple formulations, based on natural, synthetic, and hybrid polymers. Natural polymers such as hyaluronic acid (HA), collagen (Col), gelatin (Gel), or fibrinogen (Fib), exhibit superior biocompatibility, bio-degradability, and biological activity compared to synthetic ones [34,35,38], HA is a linear anionic non-sulphated high molecular weight glycosaminoglycan (GAG) with high viscoelasticity and lubricity with high water retention

[0039] , Furthermore, numerous studies have highlighted its role in wound healing by enhancing the migration and differentiation of mesenchymal and epithelial cells, as well as inducing angiogenesis and Col deposition

[0040] , Col is a structural protein that enables cell adhesion and proliferation, it has low immunogenicity and enhances fibroblast migration, which pro-motes wound healing. Considering that the main function of this protein is to provide overall strength, shape, and integrity to tissues and organs, artificial-based Col scaffolds are increasingly being considered [41 ,42], Fib is a crucial element in skin regeneration, given that throughout the coagulation cascade it is transformed into an insoluble fibrin network that induces fibroblast proliferation, migration, and the pro-duction of Col, GAGs, and proteoglycans. Moreover, the resulting matrix is biodegradable and has substantial mechanical strength

[0043] , Finally, Gel is a protein derived from Col hydrolysis. Due to its richness in arginine-glycine-aspartic acid sequences, it allows cell attachment, and it presents high adaptability for chemical modifications. However, it is a thermotensile material. These features, together with the fact that all four molecules are essential components of the ECM, make it possible to generate bio-mimetic systems that resemble the physiological environment of the skin and provide a more reliable source for regeneration [44,45],

[0218] Nevertheless, these materials are often unable to proportionate enough mechanical strength to consider the bioink appropriate for printing. Considering this fact, non-biomimetic materials, such as alginate (Alg), can be also used in order to improve the performance of the ink

[0046] , This molecule is a natural block copolymer, concretely a linear anionic polysaccharide, which can be easily crosslinked by the addition of CaCI2. Its primary function in TE applications is to provide mechanical integrity, which is why Alg hydrogels have been highly implemented for skin, bone, and cartilage regeneration applications [33,34], However, its main drawback is that it does not provide mammalian cell adhesive ligands

[0047] , This fact is solved by its hydrophilicity, water solubility, and specific functional groups, features that facilitate its mixture with other biopolymers that are able to enhance the cell attachment into the scaffold, such as Col, HA, fibrin, Gel, chondroitin sulphate (Chon), etc.

[0219] When talking about TE, the focus must not be only put on the matrix, but also on the methodology used for the synthesis of the system. Three-dimensional (3D) bioprinting is a technique that allows engineering biological constructs by sequentially dispensing a cellladen biomaterial (bioink), layer by layer by computer-aided design [48-50], The versatility of this process allows the conformation of multilayer structures, which must be biocompatible and maintain suitable properties to match the tis-sues, with different cell compositions [51 ,52], The progressive trend of this technology is developing portable handheld bioprinters, which could be used by surgeons and physicians for in situ bioprinting. Therefore, it aims to improve and accelerate healing procedures since de novo tissues are produced immediately on the predicted anatomical site in the real organism. Recent research has shown the utility and potential of handheld bioprinters notably in the areas of skin, bone, and cartilage regeneration [53-55], Currently, multiple devices have been developed, which are highly versatile, as they use a variety of bioinks, crosslinking agents, and multiple printing methodologies [56-60],

[0220] Depending on the desired applicability of the scaffold and the technique used to synthesize it, several rheological parameters must be considered, not only to get the better performance of the device, but also to ensure the maximal cell viability. Features such as the flow rate, shear stress, pressure, and viscosity are key parameters that are interconnected and have a critical role in maintaining the survival and the proper metabolism of the cells during the printing process [61 ,62], For extrusion bioprinting, the material not only should be sufficiently viscous to be dispensed but must also have sufficient strength and stiffness to maintain structural integrity after printing (yield stress). Usually, nonNewtonian fluids are more optimal for printing, since their viscosity varies according to the exerting force. More concretely, the most appropriate behaviour for cell-laden inks is shearthinning, when viscosity decreases with increasing shear rates

[0063] , This is because cell viability and proliferation in bioprinted con-structs are favoured when cells are subjected to low-stress levels

[0064] ,

[0221] Apart from extrusion, using this kind of device enables multiple biofabrication methodologies, such as spraying. Topical skin sprays have recently gained increased attention as a delivery methodology for hydrogels and cell suspensions, given the advantages of this procedure: the capability of treating large injuries over topographically challenging areas, the short application time, and the homogeneous distribution of the sprayed suspension [65,66], This has enabled that several products, both acellular and cellular, have arrived to the market. One example could be TISSEEL, a fibrin sealant delivered through an air compressor spray module

[0067] , Another remarkable product is the ReCell Spray-On Skin, a kit that enables the immediate processing of a skin sample into a cell suspension for spraying or dripping into the wound

[0068] , How-ever, as in the case of extrusion devices, further research is still needed to standardize the procedures to apply the scaffolds onto the wound, as well as the bioinks and the devices.

[0222] In this study, a hand-held multifunctional biopen device with different heads for both spraying and extrusion bioprinting has been optimized for the treatment of skin lesions. Different bioinks have been formulated, and their mechanical properties, printability, and biocompatibility have been evaluated.

[0223] 2. Results

[0224] 2.1 The hand-held multifunctional biopen

[0225] The multifunctional device was designed with the aim of supporting dual dispensing, controlled by a pneumatic system (Figure 9). Placed in the middle of the chassis, there are two On / Off buttons (one for each head) connected to electronic valves that enable the opening and closure of the airflow inlet from the air pump. The air pressure, and consequently, the material deposition rate, are controlled by a pressure regulator placed at the air pump (Figure 9a). Therefore, by increasing the air pressure, a higher shear is exerted and the material is strongly pushed out through the nozzle. Because of its dual head adapter (Figure 9b), this system allows the dispensation of two solutions either simultaneously or subsequently. There are two kinds of heads for the system, one for spraying (Figure 9c) and another for extrusion (Figure 9d). In the first case, the material is dispensed through an inner nozzle, while air is pushed through an outer nozzle. When the ink arrives at the air stream near the outer orifice of the nozzles, it is pulverized generating multiple droplets. In the second case, the material is pushed through a syringe by the pressure that is exerted by the air entrance and then, it is dispensed by the nozzle (Figure 9e). Depending on the desirable applicability and the desired diameter of the extruded filament, the nozzle can also be exchanged.

[0226] 2.1.2 Functional comparison between the hand-held device and a 3D printer

[0227] As proof of concept of the device, and more concretely of the extrusion head, the performance of the hand-held 3D biopen was compared with a conventional 3D bioprinter (CELLINK Bio X6) (Figure 10). For this aim, the same material, the Fibercoll-Flex A bioink (F-F-A, Viscofan BioEngineering), was used for both instruments, and the precision and the printability were evaluated, as well as the similarity between the proper-ties of the final scaffolds.

[0228] Printing fidelity

[0229] To determine the printing accuracy of each system, a multilayer grid model was de-signed with variable pore size, and it was then compared to the scaffolds that were made with each device (Figure 10a). The same bioink (F-F-A 3%) was used in both systems and it was verified whether the original dimensions of the design were maintained or not. In the case of the biopen-fabricated scaffold, its dimensions differed more than the 3D-bioprinted one. Furthermore, not only when printing a monolayer scaffold but also with a multiple-layer design, the standard thickness and the uniformity of the extruded filament were more constant in the second case than in the first one. However, it must be remarked that the performance of the device can be highly dependent on the intrinsic properties of the bioink, and therefore, each system requires an appropriate parameter adjustment for each material. Considering this, an Alg (9%)-Gel (6%) bi-oink was also used to replicate the same grid design with the 3D biopen (Figure 10c). As it was a stiffer bioink, it was possible to fabricate scaffolds that were more similar to the original design than the F-F-A ones.

[0230] Hydrogel diffusion

[0231] The strand diffusion and axis fusion of the hydrogel were also evaluated (Figure 10b). The main fusion points are the corners of the scaffold, as well as the intersection be-tween perpendicular strands. When analysing the scaffolds with bigger pores, there was not much difference between the two samples. The only remarkable thing would be that in the case of the 3D biopen, there was more diffusion in the interconnections be-tween the perpendicular lines of the scaffold. However, as the pore size decreased, there was more internal fusion between the strands, and in the case of our device, more hydrogel diffusion was visible in the external corners of the printed scaffold.

[0232] Printability of 3D hydrogel scaffold

[0233] Following a digital cube model (Figure 10a), two 3D scaffolds were made with each printing system (Figure 10c). The final structure had a total of 25 layers, and the final height was 15 mm. During each layer deposition, the variation in fidelity was evaluated throughout the printing process. By comparing the structures fabricated with the 3D biopen and the 3D bioprinter, it can be observed that in the first case, the printing ac-curacy is slightly better maintained, as the upper layer of the scaffold was more similar to the lower initial one. However, the layer deposition in both cases was not uniform, affecting the final resolution of the structure. Furthermore, in both cases, the corners of the structure lost the angular shape as the height increased.

[0234] 2.3 Bioink formulation and evaluation

[0235] A total of 68 combinations of different materials were evaluated to determine which bioinks could result more adequate for skin regeneration. Features such as the consistency of the material, their polymerization time and the biocompatibility of the scaffold, were crucial to determine which formulations were ap-propriate. For the spray application, a bioink composed of Fib, Dermial® (Der, Bioiberica S.A.U.) and tranexamic acid (TA) (Fib-Der-TA) was selected. For extrusion, a preexisting collagen bioink, FiberFlex N (F-F-N, Viscofan BioEngineering), was used, alone or in combination with HA or with an aldehyde-modified HA (AHA). Moreover, a mixture of Alg, Gel, and xanthan (Xan) (Alg-Gel-Xan) was also chosen for extrusion. The other combinations were not able to meet all the necessary requirements for their use as bioinks for these devices. For example, the Gel-AHA bioink generated a firm hydrogel with high water retention, but it required at least 4 hours to polymerize. Furthermore, it degraded at 37 °C. Many of the mixtures that incorporated Gel in their formulation presented the same limitation: polymerization depends on the temperature. Therefore, these bioinks polymerize at room temperature, which limits their use with the actual device (as the 3D biopen does not have a thermo-regulable head). Other materials, such as for example the ones that incorporate Alg at a 10% concentration, seem to have a good consistency for printing, but when they were polymerized, the resulting scaffold was too stiff. One last case would be the F-F-A bioink, which had good consistency and printability; however, after printing and to polymerize the mate-rial, it required a neutralization step with NaOH. This does not allow to embed cells into the bioink, as they would be damaged during the neutralization process. Thus, using F-F-A, it would only be possible to culture cells on the surface of the scaffold, which limits its applicability.

[0236] 2.4 Mechanical and rheological characterization

[0237] To determine if the selected bioinks were adequate for the device, their mechanical properties were evaluated (Figure 11). Shear viscosity was analysed to determine the capability of the materials to be either sprayed or extruded through the nozzles (Figure 11a). This parameter was evaluated under a steady shearing flow at 25 °C, and as reference, the viscosity of distilled water (0.879 ± 0.067 mPa s) and a 5% Fib solution (2.061 ± 0.603 mPa s) were also measured. As expected, the Fib solution demonstrated a Newtonian flow behaviour where the viscosity was independent of the shear-rate. Contrarily, most of the inks that were evaluated showed a shear-thinning behaviour, as the viscosity decreased as the shear-rate increased. The values of viscosity of the evaluated materials are summarized in Table 1.

[0238] Bioink Viscosity (y) (mPa s)

[0239] Fib

[0240] 2.061 ± 0.603

[0241] Fib-Der

[0242] 973 to 2.85

[0243] Fib-Der-TA 2,186 to 5.413

[0244] Alg-Gel-Xan 46,000 to 1 ,610

[0245] Alg-Gel-Xan-Der 266,333 to 2, 110

[0246] Alg-Gel-Xan-Chon 9,5526 to 1 ,026

[0247] F-F-A 271 ,33 to 164

[0248] HA 11 ,221 to 8,018

[0249] AHA 5,368 ± 1 ,333

[0250] Table 1. Summary of the viscosity values of the different bioinks that were evaluated, in the range of shear-rate of 0.099 to 800 s-1.

[0251] It was observed that the viscosity of Fib-Der and Fib-Der-TA varied significantly at low shear-rates (between 0.099 and 0.556 s-1). This shear-thinning behaviour was not only observed in the sprayed bioinks, but also in the extrusion ones (and in a more pronounced way) (Figure 11 b). In the case of the Alg-Gel-Xan ink, the addition of Der and Chon (Bioiberica S.A.U., Barcelona, Spain) produced in both cases a change in the viscosity range, which was statistically significant for Alg-Gel-Xan-Der in the shear-rate interval of 0.099 to 0.259 s-1. Regarding the collagen bioinks, the mechanical properties could only be evaluated for the F-F-A bioink, as there was not enough F-F-N material to carry on with the analyses. The only material which did not follow the shear-thinning tendency was the AHA bioink. Contrary to HA, AHA had a Newtonian behaviour with a constant viscosity of 5.368 ± 1.333 mPa s.

[0252] Furthermore, the mechanical characteristics of the hydrogels were also analysed, more concretely, the compression and the viscoelastic moduli (Figure 11c, 11d and 11e). As the evaluation was made with the aim of determining the properties of the bioinks, all the hydrogels were acellular and manually-fabricated (pipetted). Data from human skin samples was used as a reference to determine whether the performance of the hydrogels was similar or not. When comparing the Fib-Der hydrogels to the ones that also incorporated TA in their formulation, they did not show any significant difference neither in the Young’s Modulus or in the viscoelastic moduli. However, these formulations could resist less compression and had lower storage and loss moduli than the human skin samples. Regarding the Alg-Gel- Xan scaffolds, they had higher Young’s and viscoelastic moduli than the Fib formulations in all the cases. However, there were no significant differences between the mechanical properties of Alg-Gel-Xan, Alg-Gel-Xan-Der, and Alg-Gel-Xan-Chon hydrogels. Regarding the Young’s Modulus, although the Alg-Gel-Xan seemed to have the most similar value to human skin, it presented higher storage and loss moduli than the other Alg-Gel-Xan-based mixtures.

[0253] To determine if the selected bioinks were biocompatible, two main parameters were analysed: the metabolic activity of the cells and their viability in the scaffolds. This procedure was done for both printing methodologies, spraying (Figure 12) and extrusion (Figures 13 and 14).

[0254] 2.5.1 Fib-Der-TA bioink

[0255] Firstly, the variation of cell metabolic activity depending on the Der concentration was evaluated (Figure 12a). All the hydrogels were manually fabricated, and the Fib bi-oink was used as a control to determine the impact of adding Der to the formulation. As it can be observed, after the first day of culture, the addition of 2.5 mg / mL of Der already had a significant impact on the metabolic activity of the cells compared to the control, and this was enhanced even more when increasing the concentration to 5 mg / mL. Then, after 3 more days of culture, the same tendency was maintained: as the Der concentration increased, so did the metabolic activity of the cells. Therefore, the condition that should be considered as the optimal one for human dermal fibroblasts (hDFs) culture would be the one with 5 mg / mL of Der. However, this bioink was too viscous, which difficulted the spraying performance without clogging. Considering this, it was determined that the Fib-Der (2.5 mg / mL)-TA bioink was more adequate as it was more fluid and could sustain the most similar metabolic activity to the bioink with 5 mg / mL of Der (even though they were significantly different).

[0256] This bioink formulation was the one used in order to optimize the working pressure of the device (Figure 12b, 12c and 12d). In order to determine the best conditions to minimize cell damage during the biofabrication process, different spraying pressures were studied by analysing cell metabolic activity with the AlamarBlue HS assay (Figure 12). The evaluation was made using the Fib-Der-TA bioink with hDFs. Thus, the difference between the samples was the working pressure (15, 20, or 30 psi). After the first two days of culture, there were no significant differences between the three hydrogels (Figure 12b). However, when analysing the fluorescence values of the last day, the highest metabolic activity was observed on the cells that were sprayed at the lowest pressure. Considering that at day 7 the hDFs sprayed at 15 psi had both the higher metabolic activity and cell viability (Figure 12c), this pressure would be considered as the best condition. However, in this case, the device could not maintain a constant and steady flow of the sprayed material. This fact had a certain impact on the hDFs, not that much on their viability, but on their appearance in the scaffold, as it was the sample where most cells were round-shaped after 3 days of culture (Figure 12d). Therefore, 20 psi was chosen as the optimal working pressure, as it still showed high metabolic activity levels, close to those of 15 psi, and had a comparable cell viability. Moreover, as can be observed in Figure 12d, independently of the spraying pressure, after 3 days of culture, the hDFs were embedded in the scaffolds showing their typical large, flat, and elongated morphology. This was also observed after 7 days of culture, meaning that the scaffold was able not only to sustain cell viability, but also to maintain its phenotype.

[0257] 2.5.2 F-F-N bioink

[0258] The F-F-N Viscofan ink was used alone as a control, and it was compared to the hydrogels that incorporated HA (F-F-N-HA) and AHA (F-F-N-AHA) into their formulation (Figure 13). In this case, two working pressures were analysed in order to optimize the performance of the device: 15 and 30 psi (data not shown). However, 15 psi was chosen as the optimal one because at 30 psi the flow extrusion rate was too fast and difficulted the correct performance of the device. As can be observed in Figure 13a, initially, the metabolic activity of the cells in the F-F-N- AHA hydrogels was highly superior to the other samples. This correlates to the cell viability of the hDFs (Figure 13b), which was significantly higher when incorporating AHA into the formulation. This tendency in the metabolic activity was sustained until day 3 of culture where the difference between the F-F-N-HA and the F-F-N-AHA could not be considered significant anymore. From this point until the last day of culture, the activity of the hDFs in these two samples was comparable. When analysing the control, it was not until the fifth day of culture that the metabolic activity of the cells could be comparable to the other samples, being significantly different from the previous days. Regarding the cell viability (Figure 13b), on day 6 of culture, the higher values were observed in the F-F-N-HA scaffold, followed by the control F-F-N, and the lower values were for the cells embedded in the F- F-N-AHA hydrogel (although the previous day there were no significant differences in the metabolic activity of the 3 samples). Moreover, contrary to the morphology observed in the Fib-Der-TA sprayed hydrogels (Figure 12d), the hDFs were mostly round-shaped in the scaffolds analysed after both 1 and 6 days of culture (Figure 12c). The only sample that seemed to contain certain spindle-shaped cells was the F-F-N-HA formulation. It must be also remarked that the final cell viability of the hDFs was superior in the sprayed hydrogels compared to this extrusion hydrogel formulation (Figure 12c).

[0259] The biocompatibility of the material was evaluated alone or in combination with Der and Chon (Figure 14). The first combination was intended to be applied for skin re-generation, so its biocompatibility was evaluated with hDFs. In the second case, as the objective was to determine if this bioink could have an application for cartilage regeneration, the compatibility was also determined using human mesenchymal stem cells (hMSCs), which can differentiate into chondrocytes (Figure 14a). In the first case, after one day on culture, the hDFs that were in the Alg-Gel-Xan-Der bioink had higher metabolic activity than the ones that were in the Alg-Gel-Xan and Alg-Gel-Xan-Chon bio-inks. However, this tendency was not sustained as days passed. After 5 days of culture, there were no significant differences between the two compositions, and furthermore, the Alg-Gel-Xan bioink appeared to have higher viability than the one that incorporated Chon. Contrarily, in the case of the hydrogels that contained hMSCs, the tendency where the incorporation of Chon to the basic bioink enhanced a higher metabolic activity, was maintained during all the experiment. When analysing the cell viability (Figure 14b), it seemed that the addition of Der and Chon to the ink enhanced cell survival after 7 days of culture (Figure 14b). However, the high sample variation affected the statistical significance, which made the data interpretation difficult. Moreover, by observing the confocal images (Figure 14c), it can be ensured that the bioink alone or in combination with either Der or Chon, was adequate for culturing both cell lines although none of them showed a spindle-shaped morphology.

[0260] 3. Discussion

[0261] Being a complex and dynamic biological process, wound healing is still under re-search in order to develop new clinical approaches that enable the restoration of damaged or diseased skin

[0069] , More concretely, bioengineered scaffolds are currently crucial in most wound healing treatments

[0070] , However, research should not only focus on the biomaterials used to fabricate these scaffolds, but also on developing and improving the technologies used to create them. One example would be the 3D bioprinting strategy, which enables the culture of cells in 3D structures and replicates more precisely the complex multi-layered construction that conforms the human skin [71 , 72]. In view of the complexity of the 3D bioprinting methodology, the current aim is to facilitate the use of these devices and make them as accessible as possible for medical use. For this reason, special interest has grown in hand-held 3D biopens, which aim to be more manageable and easier to use while maintaining the precision and the versatility of a normal 3D bioprinter [54, 55, 58, 59], In this study, we have evaluated and optimized the performance of a multifunctional 3D biopen for its ap-plication in the skin regeneration field. Thanks to its multimodular adapter, it is possible to use two printing modalities: spraying and extrusion (separately or even in combination). Furthermore, its dual conformation enables it to work with more than one bioink at the same time. Is this versatility, precisely, which enhances the value of our device, because the currently studied or commercialized systems are aimed to perform either extrusion or spraying bioprinting, and they usually do not use more than one material [53, 73], Thus, the future perspective of the investigation is focused on de-signing multimodular 3D biopen devices, which have a broader application on the tis-sue regeneration field [74, 75],

[0262] To determine until which point the performance of our extrusion head was com-parable to a standard 3D bioprinter, the printing accuracy and printability of each de-vice were analysed and compared, using the F-F-A bioink. Both instruments could replicate the different pore-size grid designs while maintaining the defined dimensions. However, the extrusion uniformity was better for the 3D bioprinter. Indeed, as the scaffold had higher infill, the fusion between crossing strands was more visible in the 3D biopen scaffolds. When evaluating the printability, either using the 3D bioprinter or our biopen device, as the height of the scaffold increased, it could not retain the initial square shape of the base. Both methodologies fabricated a final structure that did not maintain the angular corners. In the case of our device, this could be attributed to an irregular layer deposition, as sometimes it was difficult to visually control the number of layers that were placed on each spot. Nevertheless, when using the device for covering small size lesions, the precision would not be that crucial for this application. It must be also highlighted that the 3D biopen performance was more precise when using an Alg-Gel bioink, which had a higher viscosity than the F-F-A material

[0076] , This indicates that, as with the 3D bioprinter, it is necessary to calibrate the device properly for the properties of each material. However, independently of the used bioink, considering that the 3D biopen is a hand-held device, it is highly dependent on the user's steady hand. This fact forces to the use, in some cases, of low pressures to maintain a constant velocity that maximizes the extrusion uniformity and the printing fidelity: lower flow rates enable to make more precise movements. Therefore, it must be considered that because of this, generating the same quality hydrogel will be more time consuming when using the 3D biopen rather than a 3D bioprinter.

[0263] Considering the multimodal nature of our device, this study not only evaluated one formulation, but three different bioinks. As the research group had previous experience on working with Fib hydrogels and it has been highly used for skin sprays, it was selected as an optimal material for the spraying head [77, 78], However, it was not used alone, but in combination with Der and TA. Fib scaffolds depend on enzymatic polymerization, more concretely on the action of thrombin, which transforms the fibrinogen molecule to fibrin by mimicking in vitro the final stage of the blood coagulation cascade

[0079] , The resulting molecule has bioactive cues for cell signaling and cell-matrix and cell-cell interactions; however, its biocompatibility can be enhanced when combining it with molecules like GAGs (which have key biological and structural functions in the ECM and are major components of tissues like cartilage or skin) [80, 81 , 82], For this reason, and in order to fabricate a more biomimetic structure, Der was incorporated into the mixture. Even though this bioink yields a stable structure when polymerized, cells can degrade it throughout fibrinolysis, so it is necessary to incorporate an antifibrinolytic agent that blocks the engagement of the activators of this pro-cess (in our case, TA)

[0079] , When analysing the metabolic activity of hDFs in Fib-only hydrogels and in the ones that also incorporated Der in different concentrations (1 , 2.5, and 5 mg / mL), it was observed that as the Der concentration increased, so did the metabolic activity. However, until the third day of culture, there were no clear statis-tical differences between the samples. Even though in all cases the scaffold seemed to let the nutrients and oxygen arrive to the cells, a higher metabolic activity was observed in the Fib-Der (5mg / mL) bioink. By itself, the Fib solution has low and constant viscosity (2.061 ± 0.603 mPa s), but with the addition of Der (whose composition is 67% HA), it is also possible to enhance the viscosity of the material

[0083] , However, high-viscosity solutions or rapid liquid-gel transition can cause a decrease in the flow rate or even the blockage of the nozzle

[0084] , Furthermore, when correlating the mechanical properties with the biological ones, it has been reported that the materials with a shear-thinning behaviour and quick structural recovery, tend to preserve cell viability better [62, 64], This is because the viscosity of the solution decreases with higher shear-stress, so cells are less damaged. Then, it is evidence that the material has to meet certain mechanical requirements in order to be used with the spraying head of the multifunctional device, therefore, the Fib-Der (2.5 mg / mL)-TA formulation was selected for further analyses. With a viscosity ranging from 2,186 mPa-s at 0.099 s-1 to 5.413 mPa s at 800 s-1 , the Fib-Der bioink was at the same time, more viscous than the Fib bioink and less than the Fib-Der-(5 mg / mL) bio-ink, but it still provided the most similar metabolic activity to the 5 mg / mL formulation.

[0264] By using the Fib-Der (2.5 mg / mL)-TA bioink, the best working pressure for the de-vice was determined. Although none of the evaluated pressures (15, 20, and 30 psi) showed a similar metabolic activity to the control one, this could be expected as bio-fabricating a cell-loaded hydrogel by a hand-held device implies applying a flow of compressed air to the cells. Furthermore, this process involves more parameters that must be considered, and which can have a major impact on cell integrity (the nozzle diameter, the working pressure, the distance of impact, the time that cells are exposed to the shear flow...)

[0085] , Therefore, it is necessary to study how this printing parameters can affect cell viability. Regarding the air pressure, it has been previously de-scribed that it is negatively correlated to the cell growth

[0086] , so the expected results would be that as lower pressure is applied to the cells, their activity is better maintained. However, initially, even if there were no significant differences between the three spraying pressures, the metabolic activity of the cells sprayed at 20 and 30 psi appeared to be slightly higher than at 10 psi. Moreover, the cells in these samples showed the typical fibroblast morphology, whereas the 10 psi samples contained more rounded-shaped cells. This could be related to the performance of the device, at 10 psi, it could not maintain a steady flow of the material, and more time was required in or-der to spray the total volume of bioink. This same reason would justify the absence of significant differences in the cell viability analysed after 3 days of culture. The expected tendency in the metabolic activity was not observable until the seventh day of culture, even though it did not correlate with the cell viability results. Apparent differences between the metabolic activity and the cell viability could be because, even though some cells were stained green as they were alive, their metabolism was slowed down. Thus, considering the own device performance and the viability / metabolic data that was obtained, 20 psi was considered the optimal working pressure for spraying the Fib-Der-TA bioink with the current device.

[0265] For working with the extrusion head of the 3D biopen, two different kinds of bio-inks were evaluated. The first selected material was F-F-N, a collagen-based bioink that was used alone or in combination with HA. Even though higher-pressure values were analysed, 15 psi was selected as the working pressure. This was not only because previous studies have shown a decrease in the percentage of live cells when increasing the extrusion pressure

[0067] , but also because using higher pressure than 15 psi difficulted the performance of the device using the 20G nozzle. From the biological point of view, collagen type I has the potential to facilitate cell migration, remodel new tissues, and promote wound healing, thus it is appropriate for skin regeneration applications. Furthermore, this material shows shearthinning behaviour and high print resolution [57, 61 , 68], However, to generate more biomimetic scaffolds, HA or AHA were also incorporated to the Col material. During the 4 first days, the hDFs metabolic activity was significantly superior in the scaffolds that incorporated HA or AHA rather than in the basic formulation hydrogels. This also correlates with the cell viability analysis of the first day of culture, where the highest viability is observed in the F-F-N-AHA scaffolds. Considering that HA cell receptors are able to increase fibroblast proliferation

[0081] , this would justify the enhanced metabolic activity and viability in comparison to the F-F-N-only scaffold. However, it must also be considered that HA is a molecule with high water-retention capacity. This feature means that when the shear-rate increases, the viscosity decreases and the elasticity increases, so it demonstrates a shearthinning tendency that also contributes to maintaining the cell integrity

[0087] , However, on the last day of evaluation, the previous tendency was not maintained, all three samples had a comparable metabolic activity, and the cell viability was slightly higher in the F-F-N-HA scaffold. This sudden arrest on cell proliferation could be related to the in-complete gelation of the scaffold that was observed, meaning that after some days of culture the hydrogel was no longer able to proportionate enough strength to enhance cell proliferation. As the HA is embedded into the Col matrix and the hydrogel polymerization depends only on the Col neutralization

[0089] , the used concentration of HA and AHA could be too high for the amount of Col and its molecules could be interfering with the Col fibril association. Nevertheless, the AHA modification intended to generate a self-healing structure, but when combining it with the F-F-N Col, it did not appear to have this ability. Thus, further studies should be done in order to determine if using this modified HA instead of the normal one has a higher positive impact on cell viability. As there was not enough material to evaluate the rheological properties of these three bioinks, this data is not included in the study. However, the properties of AHA in comparison with HA were evaluated. Due to the aldehyde modification, the molecular chains of the original HA are less interconnected between them, and this makes that the solubility of the AHA increases. This explains the change in the original viscosity of the HA, which turned into a Newtonian fluid behaviour (AHA showed a constant viscosity of 5.368 ± 1 .333 mPa s). As the AHA solution was much more fluid than HA, its combination with the F-F-N material was easier and generated a more homogeneous mixture. The heterogenicity of the mixture could explain the lack of complete polymerization of the F-F-N-HA hydrogel, which at the same time, would explain why hDFs were more spindle-shaped than in the other hydrogels (the scaffold was less stiff and did not exert as much physical strength to the cells). Moreover, as it was more fluid, the bioink was more easily extruded, not like in the other case. The F-F-N-HA mixture was stiffer and required a higher working pressure (40 psi), which might justify the lower cell viability of the first day. Furthermore, after 6 days of culture, all the scaffolds rendered comparable cell viability even though most of the cells were roundshaped. In order to determine if this is due to the high stiffness of the scaffold, it would be necessary to repeat this same experiment with lower F-F-N concentrations.

[0266] The second kind of bioink that was considered for extrusion was the Alg-Gel-Xan one, alone or in combination with Der and Chon. In some cases, such as for example when using HA, cell-laden constructs cannot hold the 3D structure because of poor mechanical strength

[0087] , For this reason, Alg and Xan were introduced, and even though they are not biomimetic components of the skin and lack adhesive motives that can initiate cell adhesion, they are able to improve the mechanical performance of the bioink [90, 91], Gel and GAGs were also used in order to enhance the biocompatibility of the material. Even though multiple concentrations of Alg-Gel-Xan were evaluated, the 8%, 5%, and 0.5% concentrations were selected for each biomaterial, respectively, because the resulting mixture did not gel at room temperature, which was a requirement as the current 3D biopen device does not have a thermo-regulable head. The bio-logical properties of this bioink were not only evaluated on hDFs, but also hMSCs in order to see if the mixture could have any application in the field of cartilage regeneration, given that the composition of the bioink has high potential for cartilage regeneration since Chon and dermatan sulfate, included in Der, are GAGs found in cartilage ECM

[0092] , The bioinks were extruded at a working pressure of 30 psi (the minimum pressure required to extrude as the bioink was much more viscous than the previously evaluated materials). The hydrogel polymerization completely depended on the ionic crosslinking, due to the interaction between Alg and CaCI2

[0093] , Therefore, an adequate gelation time is crucial to obtain a stable scaffold for cell culture. In this case, the bioink was polymerized for 12 minutes, but it was not enough time to ensure the complete gelation of the scaffold. For this reason, as cell culture days passed, the hydrogel was more disintegrated, and it induced high variability in the data obtained to determine the metabolic activity and cell viability. This tendency was mostly seen in the hDFs scaffolds rather than in the ones that contained hMSCs, allegedly due to their higher growth rate. During the first day of culture, the hDFs embedded in the Alg-Gel-Xan-Der bioink were slightly more active than the ones in Alg-Gel-Xan, and highly superior to the Chon mixture. However, this tendency was not maintained, and after three days of culture, there were practically no significant differences between the hDFs samples. The last day of culture, the higher metabolic activity was for the Alg-Gel-Xan scaffold, which did not incorporate any GAGs or biomolecules that could enhance the cell attachment points. Contrarily, in the case of hMSCs, after seven days of culture, the Alg-Gel-Xan-Chon mixture resulted in a higher metabolic activity than the basic bioink formulation. This correlates to the cell viability analysis, where after 7 days of culture, the Chon scaffold had enhanced the cell proliferation, and in the other case it was only maintained. Even though this biological evaluation should be repeated in order to obtain more statistically significant data, it seems that the addition of Chon to the Alg-Gel-Xan bioink fomented the cell proliferation of the hMSCs. Thus, taking this fact into account, and also the fact that Chon and dermatan sulfate has been re-porter to favour the chondrogenesis of hMSCs [92, 94], these results seem to indicate that the bioink could have a potential application for cartilage regeneration. However, it is necessary to determine the proper gelation time of the mixture and make an analysis to determine the degradation time of the scaffold. Moreover, even though the scaffold seems biologically compatible for cartilage applications, mechanically does not meet the necessary requirements (considering for example that the Young’s modulus of natural cartilage is in the range of 0.45 to 0.80 MPa, far higher than the Alg-Gel-Xan-Chon modulus)

[0095] ,

[0267] Related to the rheological analysis of the Alg-Gel-Xan, Alg-Gel-Xan-Der, and Alg-Gel-Xan- Chon scaffolds, all three bioinks showed the same viscosity pattern, meaning that the mechanical properties of the initial mixture are not modified when including Der and Chon. However, it is important to highlight that the range of viscosities of these materials is more than 1000 times the range of the Fib bioinks, which entails that higher pressure is exerted on the embedded cells of the bioink when extruding the material. Nevertheless, when analysing the Young’s Modulus, the Alg-Gel-Xan mixtures were the most similar to the human skin values. This means that the final polymerized hydrogel was mechanically more comparable to the real skin than the Fib-Der-TA scaffold. However, several studies have reported that soft hydrogels like the Fib ones can be more adaptable to the wound site and have a better performance in healing

[0096] , In fact, if the confocal images of the three bioinks are compared, the one that seems to provide a more adequate environment for hDFs is the Fib-Der-TA one. Furthermore, when comparing the cell viability of all the tested bioinks, the Fib-Der-TA hydrogels seemed to have the highest values, which could be related to both, the properties of the material or to the different working pressure. Finally, when analysing the viscoelastic moduli, all three Alg-Gel-Xan hydrogels have higher storage modulus than loss, meaning that they could be considered mainly as elastic materials (and more elastic than the human skin). This would be also the case of the Fib scaffolds; however, they have lower viscoelastic moduli than the Alg-Gel-Xan ones.

[0268] 4. Materials and Methods

[0269] 4.1 Cell culture hMSCs and hDFs were isolated from human adipose tissue and skin samples, respectively. Both cell types were collected at the Vithas Hospital Granada (Plastic Surgery Service) with informed consent and Institutional Review Board approval (ethic committee number: 0467- N-20). They were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM; Gibco) supplemented with 10% foetal bovine serum (FBS) and 1 % penicillin and streptomycin (P / S; Invitrogen). The culture was maintained at 37°C in a humidified atmosphere containing 5% CO2. The medium was changed every 2-3 days, and cells were sub-cultured when they reached approximately 80% confluence.

[0270] 4.2 Bioink formulation

[0271] 4.2.1 Fibrinogen-based bioink

[0272] The bioink, which has been previously described (Pleguezuelos-Beltran et al., 2024; unpublished), consists of Fib supplemented with a rooster comb-derived GAGs and Colbased matrix (Der, Bioiberica S.A.U., Barcelona, Spain), and TA(PCCA). The matrix is composed of HA (67%), sulphated GAGs (12%); including dermatan sulphate and Chon, and Col (10.4%). To prepare the acellular bioink, firstly bovine Fib (Sig-ma-Aldrich) was dissolved in a 0.9% NaCI solution at 37 °C to obtain a final concentration of 10 mg / mL. Then, TA was incorporated into the solution at a concentration of 1 mg / mL. Once dissolved, the solution was filter-sterilized with 0.22 pm membrane filters (Merck Millipore). Finally, previously UV-sterilized Der powder was added at a final concentration of 2.5 mg / mL. In order to incorporate the cells, hDFs were gently mixed with the bioink at a final density of 1 x 106 cells / mL. To obtain the resulting hydrogels, bovine thrombin (Sigma-Aldrich), previously diluted in a 40mM CaCI2 solution at a final concentration of 50 U / rnL, was mixed with the ink in a 10:1 (v / v) ratio. The hydrogels were let to gel for at least 5 minutes at room temperature.

[0273] 4.2.2 Collagen - hyaluronic acid-based bioink

[0274] Both, Col-only and Col-HA scaffolds were evaluated. Regarding the Col-only hydrogels, Fibercoll-Flex-N (bovine dermis collagen type I, Viscofan BioEngineering) was used as the base of the bioink, and it was prepared following the manufacturer’s instructions. Firstly, in order to homogenate the material, a syringe with F-F-N was connected to an empty eccentric syringe using a junction. Then, the content was passed from one syringe to the other 21 times, and depending on the desired final concentration of the bioink, in this case 2 wt%, an eccentric syringe was filled with the required volume. At the same time, a 1.5 M Tris-HCI buffer was adjusted to pH 7.3 and filter-sterilized through a 0.22 pm filter. Then, the F-F-N was mixed with the buffer 40 times to neutralize the ink, and finally, the hDFs solution was incorporated by mixing gently at a final concentration of 1 x 106 cells / mL. The scaffolds were printed with the 3D biopen at room temperature and polymerized after adding cell culture media at 37°C overnight. In the case of the Col-HA hydrogels, the procedure was the same, except that the neutralizing step was done by mixing the F-F-N with either native HA or aldehyde-HA (AHA) solubilized in the Tris-HCI buffer at a final concentration of 50 mg / mL.

[0275] Synthesis of aldehyde-modified HA (AHA)

[0276] Firstly, 2 g of sodium hyaluronate (HA) of low molecular weight (0.6-1 MDa) (Biosynth) were dissolved in 100 mL of phosphate-buffered saline (PBS), and then, mixed with 1 g of sodium periodate (NalO4, Sigma-Aldrich) for 5 h, at room temperature and under dark conditions. In order to stop the reaction, 2 mL of ethylene glycol (Sig-ma-Aldrich) were added to the mixture. Then, the mixture was dialysed for several days in a 14 KDa dialysis membrane until the conductivity-meter measured a voltage of 0 pS. Finally, the solution was frozen and lyophilized to obtain the modified HA (AHA).

[0277] 4.2.3 Alginate-gelatin-xanthan-based bioink

[0278] All the components were UV-sterilized for 30 minutes. Gel (from bovine skin, Sigma-Aldrich) was dissolved in PBS at a final concentration of 50 mg / mL. Then, Alg (Alginic acid sodium salt from brown algae, Sigma-Aldrich) and Xan (Dayelet) were added to this solution to reach the desired final concentrations (80 and 5 mg / ml, respectively). To these solutions, Der or Chon (Bioiberica S.A.U., Barcelona, Spain) were added at 1 mg / mL and 10 mg / mL, respectively. The mixtures were stirred at 60 °C for at least 1 h to obtain homogeneous bioinks, into which cells were incorporated (1 x 106 cells / mL). To form the final scaffolds, the materials were crosslinked with a 300 mM CaCI2 filter-sterilized solution for at least 15 min. Finally, the hydrogels were washed twice with PBS before adding the cell culture medium.

[0279] 4.3 Hand-held multifunctional biopen device: design and performance

[0280] The hand-held biopen is a portable multimodular device composed of three heads (spray, extrusion, and endoscopic). For this study, the spray and extrusion heads of the device have been tested. The system comprises a 3D-printed chassis which includes the buttons to control the air entrance for spraying and extruding, a double pneumatic head adaptable to the printing modality, and both the air and the electronic connections. The chassis was designed using the Inventor 3D CAD software (Autodesk Inc.), and 3D printed with a Raise3D Pro Plus (Raise3D, USA) printer, using acrylonitrile butadiene styrene (ABS, Raise3D), due to its good mechanical properties, durability and toughness. The pressurized air inlet, which enables the printing, comes from an air pump (HABA Trading B.V., The Netherlands) and passes through 0.22 pm membrane filters to ensure air sterility.

[0281] For spraying, the bioink and its crosslinking solution are dispensed in two independent, external, and removable containers, and they are both connected to their respective spray heads. When the air entrance is activated, due to the pressure difference, the solutions ascend through the internal tubes of the containers and are expelled by the nozzle (which has a regulable diameter).

[0282] In all assays where the spraying head was used, the angle between the device and the target surface was kept at 45°, with a separating distance of approximately 10 cm. An acetate-derived plastic cone was used as a confining funnel to focalize the sprayed material into the wells of the culture plates, and therefore, avoid volume loss.

[0283] For extrusion, the air connections function similarly, with the only difference being that the entrance is directly connected with an easy connection tube to the syringe (which contains the bioink). When the airflow is activated, it increases the internal pressure of the syringe and pushes the plunger that prevents the material from spilling through the nozzle, making it possible to extrude the bioink. The extrusion head was used with the same working angle as the spray head, but the distance between the plate and the needle was approximately 1 cm.

[0284] Before using both devices and between different experiments, the containers, syringes, internal conduits, and nozzles were carefully cleaned by rinsing with ethanol, followed by multiple PBS washes.

[0285] 4.3.1 Optimization of the working pressure

[0286] To optimize the best pressure for the spray and extrusion applications, the Ala-marBlue HS assay (Invitrogen, CA, USA) was used as an indicator to determine the metabolic activity of hDFs in the Fib-Der-TA, F-F-N, and Alg-Gel-Xan-based hydrogels. It was analysed after either spraying at different pressures (15, 20, and 30 psi), or extruding at different pressures (15 and 30 psi). After 1 , 2, 3, 4, 5, and 7 days of culture, the samples were incubated with a 10% AlamarBlue HS Solution at 37 °C for 1 h. Then, fluorescence intensity was measured at 530 / 590 nm excitation / emission wavelengths in a Glomax (Promega) microplate reader.

[0287] 4.3.2 Comparison between the extrusion 3D biopen with a 3D printer

[0288] Preparation of the Fibercoll-Flex-A (F-F-A, Viscofan BioEngineering)

[0289] The F-F-A material was prepared following the manufacturer’s instructions. Firstly, to homogenate the material, a syringe with F-F-A was connected to an empty eccentric syringe using a junction. Then, the content was passed from one syringe to the other 20 times. Then, depending on the desired final concentration of the bioink, in this case 3 wt%, an eccentric syringe was filled with the required volume. Then, following the us-er guide, it was mixed with the required amount of distilled water a total amount of 40 times in order to ensure a homogeneous solution.

[0290] Accuracy evaluation

[0291] The same two scaffold designs were printed with the extrusion bio-pen and a 3D bioprinter (CELLINK BioX6). The dimensions were 2 x 2 cm and the pore size was either 5 or 2 mm. For both systems, the same diameter nozzle (21 G) and the same bioink (F-F-A) were used. For the 3D biopen performance, a pressure of 170 kPa was used, and in the case of the CELLINK BioX6 3D printer (Cellink, Sweden), 30 kPa (with variable infill: 30 to 40 %).

[0292] Printability evaluation The same 3D scaffold design was printed with both devices. Its dimensions were 10 mm x 10 mm, with a height of 15 mm, and both the nozzle and the working pressures were maintained as in the previous section.

[0293] 4.4. Physicochemical characterization

[0294] Rheological assays were done to determine the viscosity curves of the different bioinks, as well as the compression (Young’s modulus) and shearing (viscoelastic moduli) characteristics of the hydrogels. The analysis was carried out using a torsional rheometer MCR302 (Anton Paar, Austria) and all the measures were performed in triplicate in isothermal conditions at 25 °C.

[0295] 4.4.1. Rheological characterization of the bioinks

[0296] The shear viscosity of the inks was determined using a cone-plate configuration (50 mm in diameter and 1° angle). As a reference, distilled water and NaCI 0.9% solutions were used. The samples were pre-sheared at a constant shear rate of 800 s-1 for 1 minute to remove the mechanical history of the sample. Then, they were allowed to rest for 1 minute with no shear rate applied. Finally, they were continuously sheared following a logarithmic shear rate ramp from 0.01 to 800 s-1 for 5 min, with an acquisition time of 5 seconds.

[0297] 4.4.2. Mechanical properties of hydrogels

[0298] The compression and shearing characteristics of the hydrogels were measured using a plate-plate configuration (20 mm in diameter). For this aim, the hydrogels were fabricated in a cylindrical shape of 20 mm in diameter and 5 mm in height. Each experiment comprised three intervals. In the first one, the samples were gently com-pressed by the upper tool of the rheometer at a constant approaching speed of 10 pm / s, up to a normal force of 0.1 N, to determine the Young’s modulus. In the second interval, the normal force was kept constant for 30 seconds for stabilization. Finally, in the third interval, the samples were oscillatory sheared with a logarithmically increasing strain amplitude from 0.001% to 1000%, at a strain frequency of 1 Hz and a constant normal force to determine the shear viscoelastic moduli (i.e. , storage and loss moduli).

[0299] 4.5. Biological characterization To assess the biocompatibility of the different bioinks, cell viability and metabolic activity assays were performed. The metabolic activity of hDFs and hMSCs was ana-lysed with the AlamarBlue HS assay as described previously. The LIVE / DEAD Viability / Cytotoxicity Kit (Invitrogen Inc., Grand Island, NY, USA) was used to assess the cell viability after 1 and 7 days of culture. Samples were stained with calcein AM (2 pM) and EthD-l (4 pM) solutions in PBS at 37 °C for 30 min. Then, they were observed with the confocal microscope Nikon Eclipse Ti-E A1 (Nikon Instruments Europe B.V., Amsterdam, Netherlands), by using the laser settings at 494 / 517 nm for calcein AM and 528 / 617 nm for EthD-l. Then images were analysed with Imaged (Fiji) software, quantifying live and dead cells and determining the percentage of cell viability with the following equation: 100

[0300] 4.6. Statistical analysis

[0301] All graphed data are represented as mean ± standard error of the mean (SEM). The statistical evaluation was done using GraphPad Prism 8.0.1 software. Differences between multiple groups of data were tested using a two-way ANOVA with the Normality and Lognormality Test of Shapiro- Wilk. Differences between two groups of data were tested using the two-tailed Student’s t-test for unpaired samples with Welch’s correction. Differences were considered statistically significant at p < 0.05 (*), p < 0.01 (**), and p < 0.005 (***).

[0302] 5. Conclusions

[0303] It was possible to evaluate the performance of both, the spraying and extrusion head of the hand-held 3D biopen. The extrusion device had comparable printing accuracy and printability to a standardized 3D printer. Three different kinds of bioinks have been formulated: a) Fib-Der-TA, b) F-F-N, F-F-N-HA, F-F-N-AHA and c) Alg-Gel-Xan, Alg-Gel- Xan-Der and Alg-Gel-Xan-Chon. The first was used for the spraying device, the other two were used for extrusion printing and both, mechanical and biological analysis, were made to determine their potential for skin regeneration applications. The Fib-Der-TA mixture was the one that rendered the higher metabolic activity and cell viability for hDFs compared to the F-F-N and to the Alg-Gel-Xan mixtures. However, this can be related either to the different bioink composition or to the different working pressures that were used. Talking more concretely about the F-F-N bioinks, the biological features of this material appear to improve through the addition of HA or AHA. Nevertheless, a complete rheological evaluation is required in order to determine whether their mechanical properties are adequate for wound healing. Furthermore, it is necessary to determine an HA concentration that enables a complete homogenization of the F-F-N-HA bioink, and so, a proper hydrogel polymerization. Finally, related to the Alg-Gel-Xan bioinks, it has to be done a degradation test to determine which is the best polymerization time for these hydrogels. However, they present mechanical properties more similar to the human skin than the Fib bioinks and, moreover, the addition of Chon to the mixture seems to enhance the hMSCs metabolism and viability. Therefore, due to its biological composition, Alg-Gel-Xan-Chon bioink is also considered to have a potential application for cartilage regeneration, which will require a careful balance of mechanical properties to ensure the bioink possesses the necessary strength and flexibility.

[0304] Therefore, the obtained results demonstrate the utility of the device for skin re-generation applications and its possible application in other tissue engineering fields, even though in future studies it should be optimized the performance of the extrusion system with its corresponding bioinks.

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Claims

CLAIMS1 . A system (100) for providing a bioink (200), the system comprising: a. at least two spray heads (12a, 12b), each a spray head (12a, 12b) configured to receive a syringe (10a, 10b), each syringe (10a, 10b) configured to receive one or more components of the bioink (200) and wherein each a spray head (12a, 12b) comprises a nozzle (125a, 125b), b. a housing (20), configured to comprise the at least two spray heads (12a, 12b), and comprising at least two housing nozzles (22a, 22b), each housing nozzle (22a, 22b) associated to a spray head (12a, 12b) and disposed around the spray head nozzle (125a, 125b), the housing nozzle (22a, 22b) comprising a diameter bigger than the diameter of the spray head nozzle (125a, 125b), c. a gas inlet (30) connected to the housing (20) configured to receive a gas (300), and d. a control system (40); wherein the control system comprises at least one of the following two alternatives: i. when the system is for providing a bioink (200) in the form of a spray the control system, comprises at least two valves (42a, 42b), each valve (42a, 42b) associated to a housing nozzle (22a, 22b) and configured to control the flow of the gas (300) through each housing nozzle (22a, 22b), wherein each valve (42a, 42b) is further configured to control the provision of the one or more components of the bioink (200) from each syringe (10a, 10b) through its spray head nozzle (125a, 125b), by controlling the flow of gas (300) through its housing nozzle (22a, 22b), wherein the at least two valves (42a, 42b) are configured to provide the one or more components of the bioink (200) of their respective syringe (10a, 10b) independently and wherein each spray head nozzle (125a, 125b) is completely contained within its housing nozzle (22a, 22b); orii. when the system is for providing a bioink (200) by extrusion, the control system comprises at least two actuators (43a, 43b) each actuator (43a, 43b) associated to a housing nozzle (22a, 22b), wherein each actuator (43a, 43b) is configured to control the provision of the one or more components of the bioink (200) from each syringe (10a, 10b) through its housing nozzle (22a, 22b) by extrusion; and wherein the at least two actuators (43a, 43b) are configured to provide the one or more components of the bioink (200) of their respective syringe (10a, 10b) independently.

2. A system (100) for providing a bioink (200) in the form of a spray, the system comprising: a. at least two spray heads (12a, 12b), each a spray head (12a, 12b) configured to receive a syringe (10a, 10b), each syringe (10a, 10b) configured to receive one or more components of the bioink (200) and wherein each a spray head (12a, 12b) comprises a nozzle (125a, 125b), b. a housing (20), configured to comprise the at least two spray heads (12a, 12b), and comprising at least two housing nozzles (22a, 22b), each housing nozzle (22a, 22b) associated to a spray head (12a, 12b) and disposed around the spray head nozzle (125a, 125b), the housing nozzle (22a, 22b) comprising a diameter bigger than the diameter of the spray head nozzle (125a, 125b), c. a gas inlet (30) connected to the housing (20) configured to receive a gas (300), and d. a control system (40) comprising at least two valves (42a, 42b), each valve (42a, 42b) associated to a housing nozzle (22a, 22b) and configured to control the flow of the gas (300) through each housing nozzle (22a, 22b), wherein each valve (42a, 42b) is further configured to control the provision of the one or more components of the bioink (200) from each syringe (10a, 10b) through its spray head nozzle (125a, 125b), by controlling the flow of gas (300) through its housing nozzle (22a, 22b), wherein the at least two valves (42a, 42b) are configured to provide the one or more components of the bioink (200) of their respective syringe (10a, 10b) independently; andwherein each spray head nozzle (125a, 125b) is completely contained within its housing nozzle (22a, 22b) such that the provision of the one or more components of the bioink (200) from each syringe (10a, 10b) is controlled through a venturi effect on the spray head nozzle (125a, 125b) through the flowing of gas (300) through its housing nozzle (22a, 22b)..

3. The system (100) according any one of the previous claims, wherein the system (100) system is further for providing a bioink (200) by extrusion, wherein the control system (40) further comprises at least two actuators (43a, 43b) each actuator (43a, 43b) associated to a housing nozzle (22a, 22b), wherein each actuator (43a, 43b) is configured to control the provision of the one or more components of the bioink (200) from each syringe (10a, 10b) through its housing nozzle (22a, 22b) by extrusion; and wherein the at least two actuators (43a, 43b) are configured to provide the one or more components of the bioink (200) of their respective syringe (10a, 10b) independently.

4. The system (100) according to any one of the previous claims, wherein the system (100) is configured to provide a bioink (200) comprising: a. a shear rate comprised between 0.1 and 15.000 s’1and b. a viscosity comprised between 1 and 300.000 mPa s at a shear rate of 1000 s’1; at a pressure comprised between 13.8 and 241.3 kPa.

5. The system (100) according to claim 4, wherein when the system (100) is for providing a bioink (200) in the form of a spray, the system (100) is configured to provide a bioink (200) comprising: a. a shear rate comprised between 1 and 15,000 s-1and b. a viscosity comprised between 1 and 300,000 mPa s at a shear rate of 1 ,000 s’1; at a pressure comprised between 13.8 and 241.3 kPa.

6. The system (100) according to claim 4, wherein when the system (100) is for providing a bioink (200) by extrusion, the system (100) is configured to provide a bioink (200) comprising:a. a shear rate comprised between 0.1 and 500 s1and b. a viscosity comprised between 100 and 300,000 mPa-s at a shear rate of 1 ,000 s’1; at a pressure comprised between 30 and 150 kPa.

7. The system (100) according to any one of the previous claims, wherein the system (100) further comprises an UV light (50) for photocuring the bioink (200).

8. The system (100) according to any one of the previous claims, wherein the at least two valves (42a, 42b) are configured to provide the one or more components of the bioink (200) of their respective syringe (10a, 10b) sequentially and / or simultaneously.

9. The system (100) according to any one of the previous claims, wherein each of the at least two valves (42a, 42b) is an electric valve and controlled through an actuator (44a, 44b) on the housing (20) surface.

10. The system (100) according to any one of the previous claims, wherein at least one of the at least two spray head nozzles (125a, 125b) comprises a diameter of 200pm and its associated housing nozzle (22a, 22b) comprises a diameter of 500 pm.11 . The system (100) according to any one of the previous claims, wherein the at least two syringes (10a, 10b) are disposable syringes.

12. The system (100) according to any one of the previous claims, wherein the housing (20) comprises Acrylonitrile Butadiene Styrene (ABS).

13. The system (100) according to any one of the previous claims, wherein the system (100) further comprises a manometer (35) to regulate the pressure the gas (300) is provided through the gas inlet (30) to the system (100).

14. The system (100) according to any one of the previous claims, wherein the gas (300) is provided with a pressure between 10 and 20 psi, preferably between 12 and 18 psi, more preferably 15 psi.

15. The system (100) according to any one of the previous claims, wherein the system (100) further comprises a filter (32) between the gas inlet (30) and the gas (300), preferably a membrane filter, more preferably a membrane filter of at most 0.22mm.

16. The system (100) according to any one of the previous claims, wherein the viscosity of the bioink (200) is comprised within 0.5 mPa s and 104mPa s.

17. The system (100) according to any one of the previous claims, wherein the bioink (200) is a bioink formulation comprising fibrinogen and a glycosaminoglycans (GAGs) / collagen (Col) matrix, wherein the GAGs / Col matrix comprises at least a) 60 - 75% (w / w) hyaluronic acid, and b) 5 - 20% (w / w) sulphated GAGs, and c) 1 - 5% (w / w) of collagen.

18. The system (100) according to claim 17, wherein the bioink formulation further comprises human cells, preferably human mesenchymal stem cells (hMSCs), keratinocytes and / or human dermal fibroblasts (hDFs).

19. The system (100) according to any one of claims 17 or 18, wherein the amount of fibrinogen in the bioink formulation is(i) between 1 and 100 mg / ml, preferably about 10 mg / ml, and / or(ii) between 0.1 and 10 % (w / v), preferably about 1 % (w / v).

20. The system (100) according to any one of claims 17 to 19, wherein the amount of GAGs / Col matrix in the bioink formulation is(i) between 1 and 25 mg / ml, and / or(ii) between 0.1 and 2.5% (w / v).

21. Method of providing a bioink (200) for the treatment of tissue injuries or damages, wherein the method comprises: providing at least two syringes (10a, 10b) each syringe (10a, 10b) comprising one or more components of the bioink (200) in system (100) according to any one of claims 1 to 20, and actuating one or more of the at least two valves (42a, 42b) sequentially and / or simultaneously to provide the bioink (200) in the form of a spray over the tissue to be treated and / oractuating one or more of the at least two actuators (43a, 43b) sequentially and / or simultaneously to provide the bioink (200) by extrusion over the tissue to be treated.

Images