Reinforcement and sealing structure for bioprinted tissue models and method for assembling the reinforcement and sealing structure

Abstract

A reinforcing and sealing structure for bioprinted tissue models, consisting of a casing and a sealing bioink, the casing comprising an inner module including a casing base (1) and a perforated cover (2), and an outer module including a container (3) containing a technical valve (5), an outer cover (4) and a plug (6) for the technical valve (5). The invention also relates to a method of assembling the reinforcing and sealing structure.

Description

[Technical field]

[0001] The subject of the present invention is a reinforcement and sealing structure for bioprinted tissue models, and a method for assembling said structure.

[0002] Document WO1993002635A1 discloses a chamber made of a biocompatible material for implantation in the body, the chamber containing a material that is immunologically compatible with the body, the chamber being equipped with a valve that ensures to support the life of the biological material contained therein. The document does not describe the application of sealing bioinks.

[0003] Document US5786216A describes a biocompatible capsule containing cells for transplantation, including an internal support that gives the capsule tensile strength. The document does not contain any information regarding the possible use of bioinks.

[0004] Document US20070276507A1 discloses a method for reconstructing or replacing a layered organ or tissue structure, such as the bladder, in a patient in need of such treatment, comprising the steps of providing a biocompatible synthetic or natural polymer matrix shaped to fit at least a part of the hollow organ or tissue structure in need of treatment, arranging a first cell population on or within a first surface of the polymer matrix, arranging a second cell population on a second surface of the polymer matrix, and implanting the shaped polymer structure into the patient.

[0005] Document US11051509B2 discloses a device that supports the generation and regeneration of tissues and organs, functioning on its own or within the human body. It describes the printing of biological tissue in a chamber with a flow of fluids, cells and growth factors, the chamber having at least one inlet and outlet port that allows the circulation of fluids and other biological materials. The chamber is made of biodegradable polymers, glass, plastic and / or metal and can be configured in any shape that can accommodate the tissue.

[0006] Document CN111655835A describes a hydrogel containing gelatin methacrylate, hyaluronic acid methacrylate, and optionally gelatin, collagen, fibrin / thrombin, matrigel, agarose, hyaluronic acid anthramine, gelatin tyramine, and alginate. However, this document does not contain any information on the use of a reinforcing case for the printed organ.

[0007] Document EP3146939B1 discloses an implantation tool that includes cells / a scaffold and a surrounding device. The scaffold may be a natural hydrogel containing collagen, hyaluronic acid, alginate, agarose, chitosan, fibrin, gelatin, or copolymers thereof. This device can be used for cartilage repair.

[0008] The prior art describes many applications of implant devices containing cells, tissues or medicinal substances. The chambers are provided with perforated or semi-permeable walls. However, the prior art does not provide any information on the application of a set (including a case and a bioink capable of sealing the case) to protect / ensure the integrity of bioprinted organs outside the body.

[0009] The aim of the present invention was to develop a reinforcing and sealing structure for bioprinted organs, the main element of which is a case using a special sealing bio-ink, which ensures proper conditions at each stage of printing, culturing in a bioreactor and transplantation of the organ into a patient, thus allowing to maintain sterility, ensure the proper placement of blood vessels, and ensure the tightness and strength of the organ after transplantation into the living body.

[0010] The subject of the present invention is a reinforcing and sealing structure for bioprinted tissue models, which is composed of a casing and a sealing bioink, the casing comprising an inner module including a casing base and a perforated cover, and an outer module including containers containing technological valves, an outer cover, and plugs for the technological valves.

[0011] Preferably, the casing base has markers for calibrating the printer head.

[0012] Preferably, the casing is made of a biocompatible material, preferably a biocompatible resin and / or a biocompatible polymer.

[0013] Preferably, the volume of the casing is adjusted to the volume of the bioprinted tissue model.

[0014] Preferably, there is a space of at least 1.0 mm between the bioprinted tissue model and the inner module, and a space of at least 0.5 mm between the inner and outer modules.

[0015] Preferably, the bioink includes an extracellular matrix buffer in its composition.

[0016] Preferably, a sealing bioink is used that contains an extracellular matrix, methacrylated hyaluronic acid, methacrylated gelatin, and a buffer containing a photoinitiator, preferably lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0017] Preferably, the sealing bioink comprises one part extracellular matrix having a concentration of 1-10% (w / v), one part mixture of methacrylated hyaluronic acid having a concentration of 1-3% (w / v) and methacrylated gelatin having a concentration of 5-25% (w / v), in a buffer solution to which a LAP photoinitiator has been added in an amount of less than 0.5% (w / v) as a final concentration, and the ratio of methacrylated hyaluronic acid to methacrylated gelatin in the mixture is 1:0.67.

[0018] The invention also relates to a method for assembling a reinforcing and sealing structure, said method comprising the steps of: a) filling the space between the bioprinted tissue model and the casing base with a sealing bioink; b) covering the casing base with the bioprinted tissue model with a perforated cover and pressing it to create an inner module; c) optionally, refilling the space between the bioprinted tissue model and the casing base chamber with a sealing bio-ink; d) sliding the folded inner module into the container to create an outer module; e) closing the outer module with a cover; f) optionally adding a sealing bio-ink via a technological valve; g) The process of installing plugs in technical valves. [Brief description of the drawings]

[0019] The subject matter of the invention is illustrated in the drawings, in which: FIG. [Figure 1] FIG. 1 is a perspective view of a reinforcing casing of the present invention. [Diagram 2] FIG. 2 shows the exploded elements of the casing. [Diagram 3] FIG. 3 shows a top and side view of the assembled casing. [Figure 4] Figure 4 shows a table summarizing the stability testing and evaluation of the usefulness of the selected sealing bioink formulations. The following formulations were tested: a: 10% dECM hydrogel + GelMa + HaMa, b: 10% dECM hydrogel (+ GelMa + HaMa) + 1.5% agarose 1:1, c: 5% dECM + 1.5% agarose (1:1), d: 5% dECM hydrogel (+ GelMa + HaMa) + 1.5% agarose 2:1, e: 5% dECM hydrogel (+ GelMa + HaMa) + 3% agarose 2:1, f: 1% chitosan-based hydrogel + 2% agarose, g: 5% dECM + GelMa + HaMa.

[0020] The advantage of the present invention is that the entire invention is composed of a number of designed and manufactured elements that ensure the proper performance of the tissue model bioprinting process. These elements include the casing of the bioprinted model and the sealing bioink. The casing of the model is composed of two modules: an inner module and an outer module.

[0021] Figure 1 shows a perspective view of the casing. The casing is assembled from elements in successive steps of the bioprinting process to finally generate a properly functioning tissue model. The casing base 1 of the inner module is placed in the bioprinter and filled with bioink in a specific and planned manner, with or without biological material.

[0022] Such a casing structure ensures many functions of the structure used. The inner module of the casing performs the functions of support and isolation, while the casing base 1 allows the transport of the printed tissue model. Furthermore, the casing base 1 may be provided with a marker 7 for calibrating the printer head, thereby performing a calibration function. The casing has a valve for positioning the blood vessel, ensuring its proper position and orientation with respect to the printed tissue model. Apart from this, the casing base 1 of the casing performs the function of supporting the blood vessel, providing support and having a valve that allows fixing the blood vessel with respect to the tissue model. Furthermore, the inner module is designed and manufactured in such a way that a sealing bio-ink can be introduced between the printed tissue model and the casing. A space is left between the side walls of the casing base 1 and the printed tissue model. The perforated cover 2 allows the sealing bio-ink to be introduced to surround the entire tissue model and reach all the free spaces.

[0023] The outer module performs functions related to ensuring mechanical strength, ensuring the sealing of the whole tissue model; it also has a support function since the casing allows the tissue model to be placed in the bioreactor chamber. Furthermore, by appropriately selecting the shape and arrangement of the inlet and outlet containers, the tissue model can be implanted in the recipient's body. The outer module consists of a container 3 containing at least two technical valves 5, an outer cover 4, and a plug 6.

[0024] Figure 2 shows the elements of the casing disassembled. The method of assembling the casing and sealing it with sealing ink includes filling the space between the bioprinted organ and the casing base 1 with sealing bioink. The casing base 1 with the bioprinted tissue model is then covered with a perforated cover 2 and pressed to create the inner module. The perforated cover 2 may be optionally used to refill (refill) the sealing bioink. The inner module is then slid into the container 3 to create the outer module. The outer module is closed by the cover 4. The resulting structure is then placed vertically with the outlet of the container facing upwards. The sealing bioink is then injected through the lower technical valve 5. The sealing bioink is dispensed until the sealing bioink is observed at the upper technical valve 5. The correctness of the filling must be verified, i.e. visually assessed for the presence of air bubbles. If necessary, one of the technical valves 5 of the casing is tilted upwards and the sealing bioink is refilled. Finally, the plug 6 is placed in the technical valve and pressed so that the edge of the plug 6 abuts against the wall of the casing.

[0025] The ability to ensure hermeticity is one of the more important parameters and it is the result of the sum of the solutions applied: the method of closing the casing, the method of supporting and mounting the container, the type of sealing bioink and the method of use.

[0026] The casing is made of biocompatible materials: all its elements are made of biocompatible silicone, biocompatible resins or biocompatible polymers. In addition to fulfilling the biocompatibility requirements and having suitable mechanical properties, the material must ensure the possibility of sterilization at high temperatures and / or radiation sterilization.

[0027] Biocompatible materials are characterized by their ability to function appropriately in living organisms, and must have characteristics such as being non-toxic, not affecting the body's immune system, and not causing hemolysis.

[0028] The casings can be prepared in various sizes, but the most important thing is to maintain the individual components and formulations. The size of the casing needs to be designed to provide the possibility to bioprint the tissue model in the required volume and also to provide the applicability of the sealing bioink.

[0029] The sealing bio-ink serves the relevant functions of providing suitable biological conditions for the printed tissue model: filling the space between the casing elements and the tissue model, allowing the main tissue model and the casing elements to adhere to the bio-ink, and improving the mechanical properties of the bioprinted tissue model.

[0030] One of the components of the sealing bioink is the extracellular matrix produced according to patent application WO2021014359A1. Modifications introduced into the procedure, such as the antibiotics used or small modifications in pouring, have no practical impact on the overall process carried out.

[0031] The methacrylated component (e.g. methacrylated gelatin or methacrylated hyaluronic acid) is the carrier, glycerol is the lubricant, and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) is the photoinitiator.

[0032] In the studies carried out, different types of sealing bioinks were tested. -1.5% agarose solution; -Chitosan-based hydrogels with added beta-glycerophosphate (beta-GP); -5% dECM hydrogel + GelMa + HaMa; -10% dECM hydrogel + GelMa + HaMa; -15% dECM hydrogel + GelMa + HaMa; -5% dECM hydrogel + dECM powder - + GelMa + HaMa; -10% dECM hydrogel + ECM powder - + GelMa + HaMa; -20% GelMa -10% GelMa -dECM hydrogel (+GelMa+HaMa)+agarose: - 10% dECM hydrogel (+GelMa+HaMa) + 1.5% agarose (1:1); -5% dECM hydrogel (+GelMa+HaMa) + 1.5% agarose (1:1); -5% dECM hydrogel (+GelMa+HaMa) + 1.5% agarose (2:1); -5% dECM hydrogel (+GelMa+HaMa) + 3% agarose (2:1); -Chitosan-based hydrogel + 1.5% agarose (1:1); -Chitosan-based hydrogel with beta-glycerophosphate (betaGP) + 1.5% agarose (1:1); - 1.5% agarose + cellulose (1:1 and 4:1).

[0033] The present invention is presented in the following non-limiting embodiments:

[0034] EMBODIMENT 1 Casing manufacturing technology Strength testing, including the determination of pressure limits of bioprinted tissue models, justifies the need to use sealing bio-inks and model components. The pressure limit conditions that the model must meet vary depending on the physiological pressures specific to the species and research model. Pressure tests were performed to prove the validity of the use of sealing bio-inks. Tests were performed on models with both unsealed and sealed bio-inks.

[0035] After printing was completed, the creation of the model for pressure supply strength testing was started. First, the supporting bioink was removed to unclog the vascular system of the tissue model. Then, a specially designed adapter was attached to the pancreas to allow the connection of a drain and, consequently, the flow of medium. Experiments were performed in two versions, one with the sealing bioink and one without it.

[0036] To ensure a continuous flow of liquid (optimum 50-100 ml / min), the strength test began with stabilization of the flow throughout the pancreas, as well as removal of air bubbles and residual bioink contents throughout the system. Then, a pressure increase in the system was started. The test was performed until the model was visibly leaking.

[0037] In the models without sealed bioink, the first leakage occurred near the channel inlet at pressure values ​​between 15 and 29 mmHg. Meanwhile, the models sealed with the developed bioink showed the first leakage at values ​​between 32 and 49 mmHg. A total of eight full-size models were used for the tests, thus indicating that the application of the sealed bioink is fully justified.

[0038] This was to test the sealing properties of the bioink without using a fully reinforced casing. Using the bioink and casing, the pressure can be maintained at a high level of 400mmHg.

[0039] By filling the empty space between the bionic tissue model and the reinforcement casing with a dECM-based hydrogel, a satisfactory composition of sealing biocoating could be generated, which also showed perfect adhesion to the bioink used to print the tissue model. Such a bioink could fill all the gaps between the bionic organ and the reinforcement layer, preventing delamination and leaking of the bionic tissue model.

[0040] EMBODIMENT 2 Composition of sealing bioink Varieties of bioinks with different compositions were tested. Sample results of tests performed on sealing bioinks with different compositions are shown in Figure 4. Decellularized material was obtained from a local slaughterhouse, and immediately after preparation, the pancreatic tissue was precisely cleaned of fat, large blood vessels, and connective tissue and stored in PBS solution before further handling. The whole process was carried out according to the protocol published by Klak M,Int J Mol Sci 2021;22:1-16.https: / / doi.org / 10.3390 / ijms22137005

[0041] [Table 1]

[0042] From the tests performed, the sealing bioink with the most favorable characteristics was selected: 5% dECM hydrogel + GelMa + HaMa.

[0043] The characteristics of the selected sealing bioinks are as follows: -High stability within the physiological temperature range, -No cytotoxicity; - Stably attach to the bioink that forms the mass of the bionic model, - crosslinking by light with a wavelength of 405 nm; -Low viscosity, - Remains fluid under experimental conditions and allows for sufficient penetration of the model. -High DS for methacrylates, providing stable filling. -The material is highly flexible after crosslinking and does not undergo delamination.

[0044] Embodiment 3: Crosslinking Procedure: A multi-step crosslinking was used, with prolonged exposure to light of wavelengths 405 nm or 365 nm. First, a layer was crosslinked between the bionic pancreas and the inner layer of the reinforced casing, followed by a crosslinking bioink that was applied between the previously crosslinked reinforced layer and the outer part of the casing, where a sealing bioink was introduced through a technological valve in the outer casing. Crosslinking parameters: - Wavelength 405nm or 365nm; -Crosslinking time 60-2160 seconds (optimum 1440 seconds); - 1~30mW / cm for light wavelength 405nm 2 (The optimum value is 28.5mW / cm 2 ), 1 to 15 mW / cm for a light wavelength of 365 nm 2 of power.

Claims

1. A reinforcing and sealing structure for a bioprinted tissue model, comprising a casing and sealing bioink, wherein the casing comprises an inner module including a casing base (1) and a cover (2), and an outer module having a container (3) including a technical valve (5), an outer cover (4), and a plug (6) for the technical valve (5), the cover being perforated.

2. A reinforcing and sealing structure according to claim 1, characterized in that the casing base (1) has a marker for calibrating the printer head (7).

3. A reinforcing and sealing structure according to claim 1 or 2, characterized in that the casing is made of a biocompatible material, preferably a biocompatible resin and / or a biocompatible polymer.

4. A reinforcing and sealing structure according to claim 1 or 2, characterized in that the volume of the casing is adjusted to match the volume of the bioprinted tissue model.

5. A reinforcing and sealing structure according to claim 1 or 2, characterized in that a space of 1.0 mm or more in width is provided between the bioprinted tissue model and the inner module, and a space of 0.5 mm or more in width is provided between the inner module and the outer module.

6. A reinforcing and sealing structure according to claim 1 or 2, wherein the bioink comprises an extracellular matrix buffer in its composition.

7. A reinforcing and sealing structure according to claim 1 or 2, characterized in that a buffer containing an extracellular matrix, methacrylated hyaluronic acid, methacrylated gelatin, and a photoinitiator, preferably lithium phenyl-2,4,6-trimethylbenzoyl phosphine, is used as the sealing bioink.

8. A reinforcing and sealing structure according to claim 1 or 2, characterized in that the sealing bioink comprises one part of an extracellular matrix at a concentration of 1 to 10% (w / v), one part of a mixture of methacrylated hyaluronic acid at a concentration of 1 to 3% (w / v) and methacrylated gelatin at a concentration of 5 to 25% (w / v), wherein a buffer containing a LAP photoinitiator with a final concentration of less than 0.5% (w / v) is used, and the ratio of methacrylated hyaluronic acid to methacrylated gelatin in the mixture is 1:0.67.

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