The present invention relates to a method for preparing live-cell-containing hydrogel parts, for example for use in regenerative surgery, by moulding.

This application claims the benefit of the priority of European applications EP19203847.9, filed 17 October 2019, and EP20160449.3 filed 2 March 2020, both of which are incorporated herein by reference.

Background of the Invention

Biofabrication is an emerging field in which hydrogel precursor polymers are crosslinked into stable hydrogel scaffolds to host cells. Highly specific and advanced geometries are often required to satisfy the needs of medical applications.

Casting is a method widely used to achieve specific shapes, especially with thermosensitive polymers (e.g. injection moulding). As cell-laden hydrogels are much more delicate to handle than conventional polymers used in injection moulding, methods for providing objects made of hydrogels containing live cells need to satisfy stringent requirements regarding their ability to facilitate survival and propagation of the cells.

Ionic or enzymatic crosslinked hydrogels show promise with regard to their ability to support cell-based or cell-containing implant materials; methods for obtaining such hydrogels, however, are dependent on crosslinking agents (e.g. ions) penetrating into the hydrogel precursor to crosslink it.

Currently available casting methods for ionic and enzymatic crosslinked hydrogel scaffolds present a variety of problems, mainly associated with the limited diffusion of ions, enzymes or cofactors into the mould.

In common casting applications, diffusion of ions is possible only from the top opening of the mould as other openings would lead to leakage of the material prior to setting.

The time for a specific molecule to cover a certain distance by diffusion depends on the square of the distance. Current applications of casting of ionic crosslinked hydrogels are only successful in casting small samples, as the overall diffusion distance and time for diffusion is small. Major issues, however, occur as the size of the samples increases and crosslinking times are no longer compatible with cell survival.

The viability of embedded cells in these scaffolds is also directly dependent on the diffusion of nutrients and gas exchange by diffusion. Prolonged dwelling times in the mould are therefore incompatible with cell bearing gels. Furthermore, using only one opening for the diffusion of ions leads to the generation of crosslinking gradient (the material near the opening of the mould crosslinks faster than the material further away).

Finally, the time required for the whole construct to fully crosslink increases exponentially as the diffusion of ions becomes slower as the polymer crosslinks.

Current techniques for making hydrogel shapes involve the crosslinking of an entire block of hydrogel (e.g. a cube) and cutting out the required amount of material, e.g. using a punch. Although this technique may be useful for simple shapes, it cannot be used for 3D models and involves a lot of material and cell waste (Donghwan et al., ACS Nano; DOI: 10.1021 /acsnano.8b06767).

Alternatively, moulds of different materials such as Teflon or PMMA have been used. For instance, Montaser et al. (Int. J. Biological Macromolecules 124, 802-809) show a casting technique using Teflon moulds for casting. Although Teflon is a material that can be easily autoclaved, it is expensive and requires subtractive manufacturing to be produced. This allows for more degrees of freedom with respect to the technique used by Donghwan et al. (ibid), but still has limitations in terms of shape that can be casted.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to obtain cell-compatible objects, particularly implants, by moulding in a fast and clinically applicable process.

This objective is attained by the subject-matter of the independent claims of the present specification.

Summary of the Invention

In general, three main components are necessary for the manufacture of a mould according to the invention:

1) A positive form piece, i.e. the object that is meant leave its impression in the mould (any object which has a shape that can be removed by partial tension of the surrounding gel once casted) - Examples include but are not limited to: objects created with a 3D printer using fusion deposition modelling (FDM), digital light processing (DLP), stereolithography (SLA) or pre-casted object using metal/plastic moulds.

2) A mould material: any hydrogel forming polymer (including any thermo-gelling polymer), which can be loaded with a crosslinking agent compound (e.g. an ionic or enzymatic agent or other crosslinker) - examples of the hydrogel forming polymer include but are not limited to agarose, iota or kappa carrageenan, gelatine and PEG. 3) Any hydrogel precursor solution that crosslinks by action of the crosslinking agent compound (e.g. an ionic or enzymatic agent) into a stable hydrogel (any gel that crosslinks using the crosslinking agent) - examples include but are not limited to alginate, hyaluronic acid, cellulose, chitosan, polyethylene glycol, gellan gum, carageenan, and polyacrylamide, in combination with a suitably crosslinking agent that can diffuse through the polymer network formed by the mould material.

Examples for crosslinking agents include, but are not limited to, calcium chloride (alginate, gellan gum), potassium chloride, adipic acid dihydrazide, sortase, transglutaminase, tyrosinase, sodium borate, glyoxal, glutaraldehyde, zinc, divinylsulfone, dicumyl peroxide and other agents that the skilled person can conceive of readily.

An additional hydrogel may be also used in conjunction with the three components listed above. This hydrogel may be utilised in a crosslinked or uncrosslinked form.

Brief Description of the Figures Fig. 1 shows the schematic workflow of the procedure described in Example 1.

Fig. 2 shows the schematic workflow of the procedure described in Example 2.

Fig. 3 shows the schematic workflow of the procedure described in Example 6.

Fig. 4 Schematic depicting the fabrication and infill of alginate shells.

Fig. 5 shows metamolds.

Fig. 6 Finite element modelling analyzed penetration depths of eluted calcium ions from the hydrogel mold inside the alginate solution

Fig. 7 Preliminary characterization.

Fig. 8 Mechanical characterization of tissue engineered constructs made with alginate shells.

Fig. 9 Immunohistochemical staining of 5 pm thick paraffin cross-sections of materials cultured inside alginate shells for 3 weeks.

Fig. 10 Individual samples of cell-laden materials cultured in alginate shells for 3 weeks.

Fig. 11 Shape stability of an 1.2% HA-TG, 0.3% alginate ear casted using an eluting mold.

Fig. 12 Mechanical characterization of casted ears. Fig. 13 Immunohistochemical staining of 5 mih thick paraffin cross-sections of ears cultured for 3 weeks.

Fig. 14 Storage and loss modulus of different polymers when crosslinked using an eluting mold.

Terms and definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term “hydrogel” in the context of the present specification relates to an aqueous polymer gel composition comprising 0.1 to 10.0 % (m/m) of polymer.

The term alginate in the context of the present specification refers to a group of natural polymers that consist of two monosaccharides, b-D-mannuronic acid (M) and a-L-guluronic acid (G), arranged in homopolymeric (poly-mannuronate or poly-guluronate) or heteropolymeric block structures. They can be extracted from brown seaweed and do not exert any strong immunological reaction when injected into mammalian tissues [Suzuki et al., Journal of Biomedical Materials Research. 1998;39:317-22] Alginate is fully biocompatible, FDA-compliant and used widely in tissue engineering, regenerative medicine, cell encapsulation and drug delivery. Its properties can be tuned by varying the amount of a-L- guluronic acid (G) and (1 ,4)-linked b-D-mannuronic acid (M) and by functionalization with growth factors and adhesion molecules, such as an RGD-peptide (arginylglycylaspartic acid).

The term alginate sulfate, synonymously used as “sulfated alginate” in the context of the present specification, refers to a polysaccharide consisting of b-D-mannuronic acid (M) and a- L-guluronic acid (G) organized in homo- or heteropolymeric block structures. Each monosaccharide unit contains 2 hydroxyl (-OH) groups available for sulfation. The alginate polymer can have a degree of substitution of sulfation (DSs) ranging from 0 to 2 per monomer (monosaccharide unit, either b-D-mannuronic acid or a-L-guluronic acid) or 0 to 4 per disaccharide unit (either b-D-mannuronic acid-a-L-guluronic acid, b-D-mannuronic acid- b-D- mannuronic acid or a-L-guluronic acid- a-L-guluronic acid), meaning between none and all of the available -OH groups are sulfated. The degree of sulfation dictates the physical and biological properties of the hydrogel that is composed of such a sulfated alginate polymer.

The term chondoprogenitor cell in the context of the present specification relates to a stem cell partially differentiated towards cartilage lineage.

The term chondrocyte in the context of the present specification relates to a mature cartilage cell, particularly a mature cartilage cell from articular, auricular, nasal, costal, meniscus, nucleous pulposus, disc, used from autologous or allogeneic source. Chondroprogenitor cells from these tissues and perichondrium similarly can be referred to as chondroprogenitor cells, as can be stem cells from bone marrow, cartilage, fat, and blood.

The term chondrogenic cell in the context of the present specification is used as a generic term that encompasses chondrocytes and chondroprogenitor cells and specifically relates to cells able to produce cartilage, defined by the markers collagen type II and aggrecan, which are detected by classical methods for gene expression or protein deposition analysis known to the skilled artisan.

The term voxel in the context of the present specification refers to a point in three-dimensional space, analogous to the use of the term pixel in two-dimensional space.

The term “hydrogel” in the context of the present specification refers to an aqueous polymer gel composition comprising 0.1 to 5 % (m/m) of polymer.

Detailed Description of the Invention

A first aspect of the invention relates to a method for making a hydrogel object, for example an implant used in reconstructive surgery. The method of the invention comprises the following steps:

A “positive” form piece, in other words a solid object representing the form of the object to be made by the invention’s method, is submerged into an aqueous liquid mould solution. The liquid mould solution comprises or essentially consists of an aqueous solution of a hydrogel forming polymer capable of setting to form an elastic solid at ambient temperature. The key further ingredient of the mould solution is a crosslinking agent employed to crosslink the material that gives shape to the eventual hydrogel object. Additionally, buffer components and other soluble ingredients may be present as may be required by the specifications of the object and any cells that may be present therein.

Subsequent to pouring the liquid mould solution, the liquid mould solution is allowed to set, which produces a solid mould with the positive form contained therein.

The positive form piece is then removed from the solid mould to yield an empty solid mould. This may require cutting the mould in cases where the form piece was entirely surrounded by the mould. The mould material is selected to offer a certain degree of flexibility to allow extraction of the positive form piece in cases where this is required.

The positive form piece represents the object to be generated, which can be any object desirable to be manufactured from hydrogel. The object can also be an actual organ, i.e. meniscus.

The empty solid mould is then filled with a solution of a biocompatible polymer that is capable of crosslinking in presence of the crosslinking agent previously embedded in the mould solution.

Thus, the invention according to this first aspect provides A method for making an object, and comprises the steps of: a. submerging a positive form piece into a liquid mould solution comprising a hydrogel forming polymer; b. allowing the liquid mould solution to set, to yield a solid mould; c. removing the positive form piece from the solid mould to yield an empty solid mould; d. filling the empty solid mould with a solution of a biocompatible polymer capable of crosslinking in presence of a crosslinking agent; wherein the liquid mould solution comprises the crosslinking agent.

In certain particular embodiments, the liquid mould solution comprises a gel-forming polysaccharide, a gel-forming peptide and/or a gel-forming protein. In certain embodiments, the liquid mould solution consists of water (including optional buffers), the gel-forming component (the gel-forming polysaccharide, gel-forming peptide and/or gel-forming protein) and the crosslinking agent destined to crosslink the biocompatible polymer.

In certain embodiments, the solution of a biocompatible polymer does not comprise any crosslinking agent, in other words, all the crosslinking agent needed to crosslink the biocompatible polymer is comprised in the mould component of the system.

In certain embodiments, the liquid mould solution is an aqueous solution of a thermosetting hydrogel material. There is no particular need to employ thermosetting materials, but their availability, ease of handling and relatively low cost make them an advantageous material to use for this purpose.

Thermosetting polymers do not require any additional crosslinking mechanism. They are easy to use since they can be liquified by heating them above a characteristic temperature, poured and left to settle. They are also relatively fast to settle, allowing for a fast process.

In certain embodiments, the thermosetting hydrogel material for making the mould is selected from a carrageenan, agarose, gelatin and a gum, particularly a gum selected from gellan gum, acylated and/or sulfated gellan gum, guar gum, cassia gum, konjac gum, Arabic gum, ghatti gum and locust bean gum.

An example for a gel-forming peptide or protein capable of providing a mould material is gelatine or any of its derivatives, or another protein compound derived from a structural protein such as collagen.

The liquid mould solution can essentially consist of water, the hydrogel forming polymer, the crosslinking agent and optionally, buffer components selected to correspond to any eventual buffer comprised in the biopolymer solution in order to sustain conditions keeping cells viable that may be comprised in the solution of the biocompatible polymer. In certain embodiments, the liquid mould solution comprises a gel-forming polysaccharide. A great number of suitable hydrogel-forming polymers are available. These include, without being limited to, galactomannan, kappa-carrageenan, alginate, alginate sulfate, cellulose, methylcellulose, ethylcellulose, bacterial cellulose, carrageenan, carrageenan sulfate, cellulose acetate, chitosan, chondroitin sulfate, dextran, hydroxypropyl dextran, fucan sulfate, gelatin, gellan gum, acylated and/or sulfated gellan gum, guar gum, gellan sulfate, cassia gum, konjac gum, Arabic gum, ghatti gum, locust bean gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan, pectin, starch, hydroxypropyl starch, ulvan (sulfated xylorhamnoglucuronan), xanthan gum and xanthan gum sulfate.

In certain embodiments, the liquid mould solution comprises one or several hydrogel forming polymers, particularly gel-forming polysaccharides, selected from the group consisting of agarose, galactomannan, carrageenan, alginate, alginate sulfate, cellulose, methylcellulose, ethylcellulose, bacterial cellulose, carrageenan, carrageenan sulfate, cellulose acetate, chitosan, chondroitin sulfate, collagen, dextran, dextran sulfate, hydroxypropyl dextran, fucan sulfate, fibrin, fibrinogen, gelatin, gellan gum, acylated and/or sulfated gellan gum, guar gum, gellan sulfate, cassia gum, konjac gum, Arabic gum, ghatti gum, locust bean gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan, pectin, starch, hydroxypropyl starch, ulvan (sulfated xylorhamnoglucuronan), xanthan gum, and xanthan gum sulfate.

In certain embodiments, the liquid mould solution comprises one or several hydrogel forming polymers, particularly gel-forming polysaccharides, selected from the group consisting of alginate sulfate, carrageenan sulfate, chondroitin sulfate, dextran sulfate, fucan sulfate, sulfated gellan gum, gellan sulfate, heparan sulfate, hyaluronan sulfate, ulvan (sulfated xylorhamnoglucuronan) and xanthan gum sulfate.

In certain embodiments, the liquid mould solution comprises one or several hydrogel forming polymers, particularly gel-forming polysaccharides, selected from the group consisting of agarose, carrageenan, locust bean gum, alginate, and xanthan gum. In certain embodiments, the liquid mould solution comprises one or several hydrogel forming polymers selected from the group consisting of agarose, carrageenan, and alginate.

In certain embodiments, the liquid mould solution comprises alginate sulfate characterized by a degree of sulfation of 0.1 - 1.1 per monomer. In certain particular embodiments, the liquid mould solution comprises alginate sulfate characterized by a degree of sulfation of 0.5 - 1.1 per monomer.

In certain embodiments, the liquid mould solution comprises alginate sulfate as the only gel forming polysaccharide, and the alginate sulfate is characterized by a degree of sulfation of 0.1 - 1.1 per monomer. In certain particular embodiments, the liquid mould solution comprises alginate sulfate as the only gel-forming polysaccharide, and the alginate sulfate is characterized by a degree of sulfation of 0.5 - 1.1 per monomer.

For applications in which loading of the mould with biological agents such as growth factors can play a role, the degree of sulfation is important in determining the rate of long term and sustained release. A high degree of sulfation favors more binding, however a balance needs to be found that allows the alginate sulfate to form a gel having the characteristics required by the method of the invention. k-carragean is one example of a thermosetting polymer well suited for the method of the present invention. It gels when the temperature of the solution is increased to 90°C and cooled back down to room temperature. Adding K+ or Ca+ ions increases the stiffness of the material by generating more bonds between the k-carragean molecules.

In certain embodiments, the liquid mould solution comprises a concentration of 1 % to 8% (w/v) of the gel-forming polysaccharide, particularly a thermosetting polysaccharide. In certain particular embodiments, the liquid mould solution comprises a concentration of 1% to 4% (w/v) of the gel-forming polysaccharide, particularly a thermosetting polysaccharide.

In certain embodiments, the liquid mould solution comprises a concentration of 2% to 8% (w/v) of the gel-forming polysaccharide, particularly a thermosetting polysaccharide. In certain particular embodiments, the liquid mould solution comprises a concentration of 2% to 4% (w/v) of the gel-forming polysaccharide, particularly a thermosetting polysaccharide.

In certain embodiments, the liquid mould solution comprises a concentration of 1.5% to 5% (w/v) of the gel-forming polysaccharide, particularly a thermosetting polysaccharide. In certain embodiments, the liquid mould solution comprises a concentration of 2% to 3.5% (w/v) of the gel-forming polysaccharide, particularly a thermosetting polysaccharide.

In certain embodiments, the liquid mould solution comprises kappa carrageenan and locust bean gum as the only hydrogel forming polymers at a total concentration of 3 to 5% w/v.

In certain particular embodiments, the liquid mould solution comprises kappa carrageenan and locust bean gum as the only hydrogel forming polymers, the concentration of kappa carrageenan is 2.5% to 3.5%, the concentration of locust bean gum is 0.5% to 1.5%, and the total concentration of kappa carrageenan and locust bean gum is 3 to 5% w/v.

In certain embodiments, the liquid mould solution contains agarose at a concentration of 0.75 - 2.5 % (w/v) as the only hydrogel forming polymer. In certain particular embodiments, the liquid mould solution contains low melting agarose at a concentration of 1 - 2 % (w/v) as the only thermosetting polymer. In certain embodiments, the liquid mould solution contains alginate at a concentration of 1.5 - 3.5 % (w/v) as the only hydrogel forming polymer. In certain particular embodiments, the liquid mould solution contains alginate at a concentration of 2.0 - 2.8 % (w/v) as the only thermosetting polymer.

The mould material is not limited to thermogelling materials, which however provide an easy and quick way for the production of the mould. Polymers that are ionically crosslinked (e.g. alginate) can also be used to produce the mould, but only to cast non ionically crosslinked material.

The method of the invention generally works with polymers that can form hydrogels triggered by a crosslinking agent that is released from the gel.

One general way by which virtually any materials can be crosslinked is to provide transglutaminase substrate peptides in the material, which are crosslinked upon calcium release.

In certain embodiments, the transglutaminase is provided in the precursor solution, and calcium acts as the diffusible co-factor for the enzyme liberated by the mould material.

Materials which contain vinyl sulfone (VS) groups can be crosslinked through release of dithiol crosslinking agents (including peptides flanked by cysteine).

In certain embodiments, the biocompatible polymer is selected from agarose, galactomannan, carrageenan, alginate, alginate sulfate, cellulose, methylcellulose, ethylcellulose, bacterial cellulose, carrageenan, carrageenan sulfate, cellulose acetate, chitosan, chondroitin sulfate, collagen, dextran, dextran sulfate, hydroxypropyl dextran, fucan sulfate, fibrin, fibrinogen, gelatin, gellan gum, acylated and/or sulfated gellan gum, guar gum, gellan sulfate, cassia gum, konjac gum, Arabic gum, ghatti gum, locust bean gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan, pectin, PEG-tranglutaminase, hyaluronan-transglutaminase, PEG-VS, HA-VS, poly(ethylene glycol), polyvinyl alcohol,— polyvinyl cellulose, starch, hydroxypropyl starch, ulvan (sulfated xylorhamnoglucuronan), xanthan gum, and xanthan gum sulfate; and the biocompatible polymer is different from the hydrogel forming polymer employed for obtaining the mould.

In certain particular embodiments, the biocompatible polymer is selected from alginate, alginate sulfate, PEG-tranglutaminase, hyaluronan-transglutaminase, PEG-VS, HA-VS, poly(ethylene glycol), polyvinyl alcohol, -polyvinyl cellulose, gelatin, chitosan, guar gum and gellan gum. In certain particular embodiments, the biocompatible polymer is different from the hydrogel forming polymer employed for obtaining the mould.

Typically, the hydrogel forming polymer has a concentration of 0.1 to 10.0% (w/w; all gel forming polymer concentrations given herein are understood to be given in mass percentage unless specified otherwise) in the solution that forms the mould. The most useful concentrations examined by the inventors did not exceed 6% as it starts to become very dense. In particular embodiments, the gel that forms the mould is characterized by a concentration of 0.25%-2% of hydrogel forming polymer. In certain embodiments, the crosslinking agent is an enzyme. Using enzymes as crosslinking agents requires adaption of the method to prevent the enzyme from being denatured in the thermogelling polymer, if such is employed. Material that is heated up to 80-90°C (e.g. the kappa carragean/locust gum) would be expected to denature any enzyme loaded. For enzymatic crosslinking, low melting thermopolymers such as low melting agarose (30/40 degrees) offer themselves as an alternative.

Additionally, techniques are available to protect the enzyme from heat degradation (Physical Chemistry Chemical Physics, 13(36), p.16265).

In an alternative embodiment, the co-factor or the enzyme activator is added in the mould, while the enzyme itself is present directly in the casted material. As co-factors or enzyme activators are much smaller than typical enzymes, this favours entry of the active agent into the volume of the cast object by diffusion.

In certain embodiments, the biocompatible polymer capable of crosslinking in presence of a crosslinking agent is selected from a compound given in the first column of the following table, with the crosslinking agent indicated in the second column on the same line:

The crosslinking of PEG (poly(ethylene glycol)) or hyaluronan (HA) conjugated to transglutaminase substrate peptides can be effected by providing the transglutaminase in the solution of a biocompatible polymer, and providing the essential cofactor of transglutaminase, calcium ions, in the mould. T ransglutaminases, substrates and methods of linking the substrate peptides to polymers are disclosed in WO2017191276A1 (U.S. Patent Application No. 16/098,342, incorporated by reference herein).

Sortase can be used to crosslink hydrogels from alginate, alginate sulfate, cellulose, methylcellulose, chitosan, chondroitin sulfate, dextran, dextran sulfate, gellan gum, acylated and/or sulfated gellan gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan by conjugating sortase substrate peptides on reactive groups of the polymer backbone as shown in Broguiere et al. Acta biomaterialia, 77:182-190, 2018.

Hydrogen peroxide can be used to make hydrogels from alginate, alginate sulfate, cellulose, methylcellulose, chitosan, chondroitin sulfate, dextran, dextran sulfate, gellan gum, acylated and/or sulfated gellan gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan by adding tyramine groups to the polymer backbone and combining with horse radish peroxidase as shown by Loebel et al., Carbo Poly, 115, 325-333, 2015.

DTT and cysteine-containing peptides can be used as a crosslinker to form gels from alginate, alginate sulfate, cellulose, methylcellulose, chitosan, chondroitin sulfate, dextran, dextran sulfate, gelatin, gellan gum, acylated and/or sulfated gellan gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan by adding a vinyl sulfone or norbornene group to the polymer backbone as shown by Ibrahim et al. J Biomed Mater Res A 2010 Aug;94(2):355- 70. and Lin et al. J Appl Polym Sci. 2015;132(8):41563. doi:10.1002/app.41563

Tetrazines can be used as crosslinker to form gels from alginate, alginate sulfate, cellulose, methylcellulose, chitosan, chondroitin sulfate, dextran, dextran sulfate, gelatin, gellan gum, acylated and/or sulfated gellan gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan by adding a norbornene group to the polymer backbone as shown by: Versatile click alginate hydrogels crosslinked via tetrazine-norbornene chemistry: Desai et al., Biomaterials, Volume 50, May 2015, Pages 30-37.

Photoinitiators can be used to initiate the photocrosslinking to form gels from alginate, alginate sulfate, cellulose, methylcellulose, chitosan, chondroitin sulfate, dextran, dextran sulfate, gelatin, gellan gum, acylated and/or sulfated gellan gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan by adding a light activatable group to the polymer backbone such as a methacrylate or acrylate group as shown by Kessel et al., Adv Sci, vol 7(18); September 23, 2020

Adipic acid dihydrazide (ADH) can be used to initiate the crosslinking to form gels from alginate, alginate sulfate, cellulose, methylcellulose, chitosan, chondroitin sulfate, dextran, dextran sulfate, gelatin, gellan gum, acylated and/or sulfated gellan gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, and mannuronan by introducing aldehyde functional groups to the polymer backbone, i.e. by oxidation by sodium periodate, as shown by Su et al: Journal of Biomaterials Science, Polymer Edition Volume 22, 2011 - Issue 13.

Calcium ions can be used to crosslink hydrogels from alginate, alginate sulfate, cellulose, methylcellulose, chitosan, chondroitin sulfate, dextran, dextran sulfate, gellan gum, acylated and/or sulfated gellan gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan by activating transglutaminase and initiating crosslinking of substrate peptides on reactive groups of the polymer backbone in the presence of transglutaminase as shown in Broguiere et al., ACS Biomater. Sci. Eng., 2:2176-2184, 2016.

Transglutaminase can be used to crosslink hydrogels from alginate, alginate sulfate, cellulose, methylcellulose, chitosan, chondroitin sulfate, dextran, dextran sulfate, gelatin, gellan gum, acylated and/or sulfated gellan gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan by initiating crosslinking of transglutaminase substrate peptides on reactive groups of the polymer backbone as shown in Broguiereet al., ACS Biomater. Sci. Eng., 2:2176-2184, 2016.

Tryosinase can be used to crosslink hydrogels from alginate, alginate sulfate, cellulose, methylcellulose, chitosan, chondroitin sulfate, dextran, dextran sulfate, gellan gum, acylated and/or sulfated gellan gum, heparin, heparan sulfate, hyaluronan, hyaluronan sulfate, mannuronan by conjugating tryamine groups on the polymer backbone as shown in Ozturk et al., Biomedical Materials, Volume 15, Number 4, June 2020.

Calcium ions can be used to crosslink hydrogels from alginate and alginate sulfate as shown in Ozturk et al., Adv Funct Mat, 26: 3649-3662, 2016. Potassium ions can be used to crosslink hydrogels from carrageenan as shown in Wen et al. (2014) J. Appl. Polym. Sci., 131 , 40975.

In certain embodiments, the liquid mould solution comprises 10 to 300 mMol/L of said crosslinking agent. In certain embodiments, the liquid mould solution comprises 50 to 180 mMol/L of said crosslinking agent. In certain embodiments, the liquid mould solution comprises -100 mMol/L of said crosslinking agent.

In certain embodiments, the crosslinking agent comprises an agent selected from calcium ions, potassium ions, adipic acid dihydrazide, sortase, transglutaminase, horse radish peroxidase, tyrosinase, sodium borate, glyoxal, glutaraldehyde, zinc, divinylsulfone, dicumyl peroxide, dithiothreitol, cysteine-containing peptides, tetrazine, photoinitiator, and hydrogen peroxide. In certain embodiments, the crosslinking agent comprises calcium ions. In certain embodiments, the crosslinking agent is selected from CaCh, CaC03, CaC204, Ca(C2H302)2, Cab, CaSC . In certain embodiments, the crosslinking agent is selected from CaC and CaC0 ₃ .

In certain embodiments, the mould material easily forms a gel without further modification of the polymer (like agarose, carrageenan, locust bean gum, gelatin). In certain embodiments, the mould material forms a hydrogel which is highly hydrated (95% water and higher) and thus is able to release the crosslinker to the filler material. The filler material (biocompatible polymer) just needs to be a liquid precursor, which starts to crosslink as the crosslinkers is released from the mould. Synthetic polymers (like PEG) as filler material work just as well as natural ones.

In certain embodiments, the positive form piece is characterized by a volume of 0.5 to 200 cm ³ .

In certain embodiments, the maximum diffusion distance from any volume voxel of the object to a point on the surface of the casted object is less than (<) 5 cm.

In certain embodiments, the system is designed so that a thickness of mould around the object: 20% of the object size is achieved.

In certain embodiments, the solution of a biocompatible polymer comprises live cells. In certain particular embodiments, the cells are selected from stem cells, chondrocytes and/or chondroprogenitor cells. In certain particular embodiments, the cell concentration ranges from 1 E4 (1x10 ⁴ ) cells to 5E8 cells/ml, more particularly from 1 E6 cells/ml to 1 E7 cells/ml.

In certain embodiments, the object is cast in the mould, the object is formed by crosslinking of the solution of a biocompatible polymer and then the object is transferred into culture media. Alternatively, the entire system (casted object + mould) is placed in culture media. In any of these embodiments, the casted object may contain cells that can grow in a confined space to achieve a defined geometry.

In certain embodiments, the liquid mould solution comprises 2 - 8% (w/w) of a gel forming polymer, particularly 3 to 6%, more particularly 3.5% to 4.5% or 3.5% to 5.0%, even more particularly approximately 4% of a gel forming polymer.

In certain embodiments, the solution of a biocompatible polymer from which the resulting object is formed, comprises 0.5-4% (w/w), particularly 0.5 to 2.0% of a crosslinkable biocompatible polymer.

In certain embodiments, the ratio of the concentration of the hydrogel forming polymer in the liquid mould solution to the concentration of the biocompatible polymer in the solution of a biocompatible polymer used for making the object is in the range of 2 - 5.

The density of the material used for the mould should allow it to be handled and to retain its shape when placed on a flat surface.

In certain embodiments, the positive form represents a substrate covered by a cell-containing implant layer, and in the moulding step, a substrate piece is covered by the solution of a biocompatible polymer.

The invention comprises of the making and use of a mould based on a hydrogel material, which elutes a crosslinking agent to improve the crosslinking of the casted material. The inventors envision these hydrogel shapes to have use particularly for soft tissue implants where their form contributes to the aesthetics of the face. Previously it has not been possible to cast large and complex shapes, due to the difficulty in achieving uniform crosslinking.

The invention allows for crosslinking of complex hydrogel shapes which are uniformly crosslinked through diffusion of ions or enzymes from all sides.

This approach is universal and can be used with any pourable material.

This technique allows to reduce up to 25 times the time required for crosslinking and allows cells to be embedded in large structures while reducing cell death to a minimum.

The following components are required:

1) An object (can be 3D printed) which has the form of the final desired hydrogel shape

2) A container to hold the 3D object

3) A gelling mould material which is sufficiently elastic so that the 3D object can be removed from the mould without tearing;

4) A hydrogel precursor solution which is crosslinked by presence of ions or enzymes. Stability of the mould is important. If it deforms after pouring then there is not good fidelity between the original object and that cast.

The mould material contains ions or enzymes which can freely elute out. The 3D object is placed into the container and the moulding material is poured around the object and left to solidify. Once the 3D object is removed, an impression is left in the mould (the negative mould). An ionically or enzymatically crosslinkable hydrogel is then cast into the mould and allowed to crosslink for a few minutes up to an hour, depending on the size. A schematic of this process is shown in Fig. 1.

The invention thus provides a fast and inexpensive hydrogel casting system, which elutes molecules to crosslink hydrogels with increased speed, efficiency, shape fidelity and biocompatibility.

Technical improvements provided by the invention include:

1 ) Ease of manufacture - the method consists of a few easy steps, which can be easily scaled up. All steps can be conducted with readily available lab equipment and all necessary materials are cheap and easy to handle.

2) Uniformity in crosslinking - the method of the invention allows to crosslink hydrogel precursors uniformly without any generation of gradients and therefore resolves the most common issues in hydrogel moulding.

3) The method can be tuned to adapt to requirements concerning crosslinking time and molarity. Changing the casting material and the molarity of the ions loaded in the mould gel makes it possible to increase or reduce the time required for crosslinking of the casted object.

4) Freedom of shape of casted object - given the flexibility of the moulding material, it is possible to cast shapes with large overhangs or detailed features, which is not possible with current casting techniques.

5) Reproducibility -owed to the ease of handling and the quality of the products, it is possible to replicate this procedure easily without any variation in shape at high consistency, facilitating that clinical QM process requirements are met.

Depending on an object’s shape, it is hard to remove the cast objects from a mould. The invention’s moulds are destructible, meaning that they can be stretched or, if needed, torn and broken in a gentle way, which does not injury the delicate cast object.

As stated above, the viability of scaffolds embedded cells depends on the diffusion of nutrients and gas into the gel. Prolonged dwelling times in the mould are therefore incompatible with cell bearing gels. The present invention method drastically reduced the overall time required for the crosslinking (while making it uniform). Whilst the process is still diffusion limited, the limits of in terms of the implant’s size and shape, and in terms of what cells can be embedded are greatly expanded by the invention.

As an alternative, the casted hydrogel can be allowed to crosslink only for a few seconds or minutes. The un-crosslinked polymer leftover can be removed thereby generating a thin hydrogel ‘sleeve’ that covers the surrounding of the mould. The space within the sleeve can then be filled with an additional cell-laden hydrogel solution, crosslinked or not, provided its viscosity is high enough to prevent cell sedimentation. The sleeve allows to support the shape of the graft, even if the internal hydrogel has little or no mechanical stability. At the same time, the sleeve provides a semi-permeable membrane for ions and molecules to diffuse. Molecules of small size are allowed to be exchanged but larger molecules are slowed down or blocked. The membrane’s permeability is dictated by the sleeve polymer type and content, where larger polymer molecule size and higher polymer density reduce molecule diffusion. The sleeve can be later on removed during culture once the internal hydrogel has been remodelled into a stable matrix by cells. A schematic of this process is shown in Fig. 3.

Wherever alternatives for single separable features such as, for example, a liquid mould solution material, a biopolymer or a cell type, are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

Items

The invention further encompasses the following items, without being limited to them:

Item 1. A method for making an object, said method comprising the steps of: a. submerging a positive form piece into a liquid mould solution comprising a hydrogel forming polymer; b. allowing said liquid mould solution to set, to yield a solid mould; c. removing said positive form piece from said solid mould to yield an empty solid mould; d. filling said empty solid mould with a solution of a biocompatible polymer capable of crosslinking in presence of a crosslinking agent; characterized in that said liquid mould solution comprises said crosslinking agent.

Item 2. The method according to item 1 , wherein the liquid mould solution is an aqueous solution of a thermosetting hydrogel material. Item 3. The method according to item 1 or 2, wherein the liquid mould solution comprises a gel-forming polysaccharide, particularly one or several selected from the group consisting of agarose, galactomannan, (kappa-) carrageenan, alginate, alginate sulfate, cellulose, methylcellulose, ethylcellulose, bacterial cellulose, carrageenan, carrageenan sulfate, cellulose acetate, chitosan, chondroitin sulfate, collagen, dextran, dextran sulfate, hydroxypropyl dextran, fucan sulfate, fibrin, fibrinogen, gelatin, gellan gum, acylated and/or sulfated gellan gum, guar gum, gellan sulfate, cassia gum, konjac gum, Arabic gum, ghatti gum, locust bean gum, heparin, heparan sulfate, hyaluronan, hyaluronan tyramine, hyaluronan sulfate, mannuronan, pectin, starch, hydroxypropyl starch, ulvan (sulfated xylorhamnoglucuronan), xanthan gum, and xanthan gum sulfate.

Item 4. The method according to item 1 or 2, wherein the liquid mould solution comprises agarose, (kappa-) carrageenan, and or a gum selected from gellan gum, acylated and/or sulfated gellan gum, guar gum, cassia gum, konjac gum, Arabic gum, ghatti gum and locust bean gum as the hydrogel forming polymer.

Item 5. The method according to any one of the preceding items, wherein the liquid mould solution comprises 1-4% (w/w), particularly 2-4%, more particularly 2.5 - 3.5% of the hydrogel forming polymer.

Item 6. The method according to any one of the preceding items, wherein the liquid mould solution comprises 1-8% (w/w), particularly 2-8%, more particularly 1.5 - 5% of the hydrogel forming polymer.

Item 7. The method according to any one of the preceding items 1-6, wherein the liquid mould solution comprises kappa carrageenan and locust bean gum as the only hydrogel forming polymers at a total concentration of 3 to 5% (w/v), particularly wherein the concentration of kappa carrageenan is 2.5% to 3.5%, the concentration of locust bean gum is 0.5% to 1.5%.

Item 8. The method according to any one of the preceding items 1-6, wherein the liquid mould solution comprises agarose at a concentration of 0.75 - 2.5 % (w/v) as the only hydrogel forming polymer, particularly at a concentration of 1 - 2 % (w/v).

Item 9. The method according to any one of the preceding items 1-6, wherein the liquid mould solution comprises alginate at a concentration of 1.5 - 3.5 % (w/v) as the only hydrogel forming polymer, particularly at a concentration of 2.0 - 2.8 % (w/v). Item 10. The method according to any one of the preceding items, wherein the biocompatible polymer is selected from agarose, galactomannan, carrageenan, alginate, alginate sulfate, cellulose, methylcellulose, ethylcellulose, bacterial cellulose, carrageenan, carrageenan sulfate, cellulose acetate, chitosan, chondroitin sulfate, collagen, dextran, dextran sulfate, hydroxypropyl dextran, fucan sulfate, fibrin, fibrinogen, gelatin, gellan gum, acylated and/or sulfated gellan gum, guar gum, gellan sulfate, cassia gum, konjac gum, Arabic gum, ghatti gum, locust bean gum, heparin, heparan sulfate, hyaluronan, hyaluronan tyramine, hyaluronan sulfate, mannuronan, pectin, PEG-tranglutaminase, hyaluronan-transglutaminase, PEG-VS, HA-VS, poly(ethylene glycol), polyvinyl alcohol, -polyvinyl cellulose, starch, hydroxypropyl starch, ulvan (sulfated xylorhamnoglucuronan), xanthan gum, and xanthan gum sulfate; particularly wherein the biocompatible polymer is selected from alginate, alginate sulfate, PEG-tranglutaminase, hyaluronan-transglutaminase, hyaluronan tyramine, PEG-VS, HA-VS, poly(ethylene glycol), polyvinyl alcohol, polyvinyl cellulose, gelatin, chitosan, guar gum and gellan gum; and wherein the biocompatible polymer is different from the hydrogel forming polymer employed for obtaining the mould.

Item 11. The method according to any one of the preceding items, wherein the biocompatible polymer is selected from alginate, hyaluronan, hyaluronan tyramine, hyaluronan sulfate, and hyaluronan-transglutaminase,

Item 12. The method according to any one of the preceding items, wherein the solution of a biocompatible polymer comprises alginate, particularly wherein the solution of a biocompatible polymer comprises alginate as the only crosslinkable polymer.

Item 13. The method according to any one of the preceding items, wherein the solution of a biocompatible polymer comprises alginate, particularly wherein the solution of a biocompatible polymer comprises alginate as the only crosslinkable polymer.

Item 14. The method according to any one of the preceding items, wherein the solution of a biocompatible polymer comprises 0.5-6% (w/w), particularly 0.5 to 2.0% of the biocompatible polymer capable of crosslinking in presence of a crosslinking agent. Item 15. The method according to any one of the preceding items, wherein the ratio of the concentration of the hydrogel forming polymer in the liquid mould solution to the concentration of the biocompatible polymer is in the range of 2 - 5.

Item 16. The method according to any one of the preceding items, wherein the solution of a biocompatible polymer comprises live cells.

Item 17. The method according to any one of the preceding items, wherein a. said empty solid mould is filled with a first biocompatible polymer solution capable of crosslinking in presence of a crosslinking agent, and said first biocompatible polymer solution is allowed to crosslink only for a time sufficient for the crosslinking agent to permeate and crosslink a fraction of the mould; subsequently, b. a fraction of the first biocompatible polymer solution is not crosslinked, and is removed from the mould, leaving a mould covered with a layer of crosslinked first biocompatible polymer, and c. the mould covered with a layer of crosslinked first biocompatible polymer is filled with a second biocompatible polymer solution comprising cells.

Item 18. The method according to Item 17, wherein the second biocompatible polymer solution is capable of crosslinking in presence of the crosslinking agent capable of crosslinking the first biocompatible polymer solution.

Item 19. The method according to Item 17 or Item 18, wherein said layer of crosslinked first biocompatible polymer is characterized by a thickness of 1 mm to 25 mm, particularly by a thickness of 3 mm to 15 mm.

Item 20. The method according to any one of the preceding items, wherein the positive form piece is characterized by a. a volume of 0.5 to 200 cm ³ and/or b. the maximum diffusion distance from any volume voxel of the object to a point on the surface of the casted object being less than (<) 5 cm.

Item 21. The method according to Item 16 to Item 20, wherein the cells are selected from stem cells, chondrocytes, progenitor cells chondrogenic cells, osteoblasts, endothelial cells, hepatocytes, fibroblasts and/or neurons.

Item 22. The method according to any one of Item 16 to 13, wherein the cells are present in a concentration ranging in the solution of a biocompatible polymer from 1 E4 cells to 5E8 cells/ml, particularly from 1 E6 cells/ml to 1 E7 cells/ml. Item 23. The method according to any one of the preceding items, wherein the positive form represents a substrate covered by a cell-containing implant layer, and in the moulding step, a substrate piece is covered by the solution of a biocompatible polymer.

Item 24. An object comprising or essentially consisting of hydrogel, optionally including live mammalian cells, obtained by a method according to any one of the preceding items.

Item 25. The object according to item 24, wherein the object is a medical implant.

Item 26. A kit for practicing the method according to any one of items 1 to 23, comprising a. A first component comprising or essentially consisting of a thermosetting hydrogel material, particularly in dry form; b. A crosslinking agent, wherein the crosslinking agent is present in the first component or wherein the crosslinking agent is provided in a form capable of being combined with the first component and water, to form a liquid mould solution, and c. A second component comprising or essentially consisting of a biocompatible polymer, particularly in dry form, wherein the biocompatible polymer is capable of being crosslinked by said crosslinking agent.

Item 27. The kit according to item 26, wherein the first component is provided in a first container configured to receive a first amount of water to form a first mould solution; and wherein the second component is provided in a second container configured to receive a second amount of water to form a polymer solution.

The containers can be marked in a way to allow easy proportioning of water or buffer into the containers in accordance to the amount of hydrogel mould material and implant forming biocompatible polymer, respectively. The second component is free of crosslinking agent. This allows for a fast combination of all components to make implants from a form piece derived from individual human forms as provided, for example, by rapid prototyping / 3D printing on the basis of computer generated models of the desired implant.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope. Figures

Fig. 1 shows the schematic workflow of the procedure described in Example 1 as an exemplary embodiment of the process of the invention. A1) organ model made by additive manufacturing (DLP); here: ear; A2) container; A3) mould solution;

A4) crosslinking agent; A5) gelled mould; A6) syringe loaded with biocompatible polymer and cells; A7) casted polymer; A8) cells; A9) cross-linking agent diffusing out of mould and into biocompatible polymer; A10) cross-linked, cell-laden implant removed from mould.

Fig. 2 shows the schematic workflow of the procedure described in Example 2 as an exemplary embodiment of the process of the invention. B1) organ model inflated by 1mm fabricated using additive manufacturing (DLP); here: porous graft in the shape of an orthopaedic implant; B2) container; B3) mould solution; B4) crosslinking agent; B5) gelled mould; B6) organ model - same as in (B1) but not inflated - to be casted; B7) syringe loaded with biocompatible polymer and cells; B8) cells and biopolymer poured between mould and implant; B9) cells; B10) cross-linking agent diffusing out of mould and into biocompatible polymer; B11) cross-linked, cell-laden polymer+cell layer crosslinked to implant removed from mould.

Fig. 3 shows the schematic workflow of the procedure described in Example 6 as an exemplary embodiment of the process of the invention. C1) organ model made by additive manufacturing (DLP); here: ear; C2) container; C3) mould solution;

C4) crosslinking agent; C5) gelled mould; C6) syringe loaded with polymer to generate outer shell; C7) injected polymer to generate outer shell; C8) cross-linking agent diffusing out of mould and into biocompatible polymer; C9) crosslinked layer of polymer i.e. crosslinked protective shell; C10) syringe used to aspirate the outer shell’s uncrosslinked polymer; C11) syringe loaded with biocompatible polymer and cells; C12) injected polymer and cells; C13) cells; C14) uncrosslinked, viscous cell laden ear shaped implant wrapped in a thin crosslinked-polymer-layer removed from mould.

Fig. 4 Schematic depicting the fabrication and infill of alginate shells: A) alginate solution is poured inside a ion-eluting mold B) ions diffuse into the alginate solution C) a thin shell of crosslinked alginate is formed and non-crosslinked alginate is removed D-E) alginate shell can be filled with any material F-G) the top opening is covered with more, un-crosslinked alginate and covered with ion-eluting hydrogel H) alginate shell completely encloses the infilled material. Fig. 5 Metamolds: A) Top part of the metamold for an ear cartilage model B) Bottom part of the metamold for an ear cartilage model C) Two metamolds combined for the casting of an ear cartilage model.

Fig. 6 Finite element modelling analyzed penetration depths of eluted calcium ions from the hydrogel mold inside the alginate solution. The penetration depth was extracted from the FEA simulation and reported as a scatter plot. A characteristic logarithmic skewed penetration depth curve can be observed as 100 mM CaCI2 ions diffuse in a 5mm disc composed of an aqueous solution.

Fig. 7 Preliminary characterization (A) Thickness evaluation of alginate shells generated using different polymer concentrations (0.5% w/v, 1% w/v and 2% w/v) and left to crosslink for different time lengths (20s, 60s and 180s). FEA simulation to predict the diffusion distance of 100 mM CaCI2 solution in water was used to predict the crosslinking distance in alginate (orange curve). (B) Thickness evaluation of samples after 1 week of culture; the alginate shell is stable for all conditions and supports internal material swelling without rupturing.

Fig. 8 Mechanical characterization of tissue engineered constructs made with alginate shells. Samples were cultured for 3 weeks in chondrogenic media, cut in the transverse plane and probed at 3 different sections.

Fig. 9 Immunohistochemical staining of 5 pm thick paraffin cross-sections of materials cultured inside alginate shells for 3 weeks. Scale bar corresponds to 100 pm.

Fig. 10 Individual samples of cell-laden materials cultured in alginate shells for 3 weeks.

Fig. 11 Shape stability of an 1.2% HA-TG, 0.3% Alginate ear casted using an Eluting

Mold. A and B) HATG-Alginate casted ears, C) Original 3D printed ear model used to generate the Eluting Mold.

Fig. 12 Mechanical characterisation of casted ears using a Bioindenter (Anton Paar).

Different locations on each ear are probed. The stiffness at each location is reported as a heatmap for both ears.

Fig. 13 Immunohistochemical staining of 5 pm thick paraffin cross-sections of ears cultured for 3 weeks. Scale bar corresponds to 200 pm.

Fig. 14 Storage and loss modulus of different polymers when crosslinked using an eluting mould. A) 1% Alginate crosslinked using a 100 mM CaCh loaded eluting mould. B) 0.5% Alginate-Sulfate crosslinked using a 100 mM CaCh loaded eluting mould. C) 1% Gelatin-Tyramin crosslinked using a 0.1% H2O2 loaded eluting mould. D) 2.2% HA-Tyramin crosslinked using a 0.1% H2O2 loaded eluting mould. Examples

One exemplary embodiment is given in the following:

Example 1 ( see Fig. 1):

1) 3D print an ear model using a DLP printer;

2) Prepare an aqueous solution of kappa carrageenan 3 w/v %, locust bean gum 1 w/v % and 100 mMol/L CaCh and heat the solution to 80°C until complete solution of the polymer;

3) Place and secure the 3D printed ear model inside a container with width and height at least as large as the ear model with a minimum of 10 mm of clearance on each side;

4) Pour the kappa carrageenan and locust bean gum solution in the mould and leave to cool down to room temperature;

5) Remove the ear model from the gelled mould (thereby creating an CaCh eluting mould);

6) Pour 1% alginate solution containing 10 million chondrocytes per ml in the mould and cover the top using a filter paper soaked in 100 mMol/L CaCh 7) Wait 15 minutes to allow the ions to diffuse into the casted alginate, form an initial shell, and separate the alginate ear from the mould

8) A cell-laden ear shaped scaffold with high precision to the original model is created. Example 2:

Process to cast chondrocytes and hyaluronan gel onto a porous graft, such as bone or 3D printed mineralized structures.

The process is based on a hydrogel mold which elutes Ca ²⁺ ions to crosslink the hyaluronan gel and chondrocyte solution, thus creating a thin cartilaginous layer on the porous implant (see Fig. 2)

Components: 1) Porous grafts in the shape of an orthopedic implant (minus the cartilage layer)

2) Molding device 1) A thermo-gelling mold material (i.e. kappa carrageenan 3 % w/v, locust bean gum 1% w/v and 100 mMol/L CaCh)

3) 1.5% hyaluronan gel, 0.25% alginate, 10x10 ⁶ chondrocytes/ml Procedure:

A template of the entire implant (Bone + Cartilage layer) is 3D printed from medical grade polymer. The molding solution is heated to 80°C and poured into the container. The template is mounted onto the holder and inserted into the container until the mold material has set (30 min at 4°C). The template is then removed, leaving a negative imprint.

The bone graft is mounted onto the second holder and lowered into the container. Using the inlets, the precursor cell solution is injected into the gap left between the eluting mold and the porous bone graft using a 2ml syringe. After 30 min the precursor solution has set and the graft is removed.

Alternative Example 2:

1) Obtain an MRI/CT scan from a patient with a nose defect

2) Reconstruct the nose shape by generating a 3D model

3) Print the model using a stereolithographic printer using a UV curable resin

4) Prepare an aqueous solution of low melting 2 w/v % agarose with 100 mMol/L CaCh and heat the solution to 40°C until complete solution of the polymer;

5) Place and secure the 3D printed nose model inside a container with width and height at least as large as the ear model with a minimum of 10 mm of clearance on each side

6) Pour the agarose solution in the mould and leave it to cool down to room temperature;

7) Remove the nose model from the gelled mould (thereby creating an CaCh eluting mould);

8) Pour 1% alginate solution containing 5 million chondrocytes per ml in the mould and cover the top using a filter paper soaked in 100 mMol/L CaCh

9) Wait 15 minutes to allow the ions to diffuse into the casted alginate, form an initial shell, and separate the alginate nose from the mould

10) A cell-laden nose shaped scaffold with high precision to the original model is created.

Example 3:

1) Obtain an MRI/CT scan of a knee from a patient requiring patella replacement

2) Extract from the acquired data the shape of the patella (knee cap)

3) Print the knee cap model using an SLS printer 4) Prepare an aqueous solution of low melting 2 w/v % agarose with 100 mM CaCh and heat the solution to 40°C until complete solution of the polymer;

5) Place and secure the 3D printed knee cap model with dimensions increased by 10% inside a container with width and height at least as large as the knee model with a minimum of 10 mm of clearance on each side

6) Pour the agarose solution in the mould and leave to cool down to room temperature;

7) Remove the knee cap model from the gelled mould (thereby creating an CaCh eluting mould);

8) Place the knee cap model at 100% size and secure it at equal distance from all sides. This will generate a gap between the mould and the knee cap of 1mm.

9) Pour 1 w/v % hyaluronic acid transglutaminase (HATG) solution containing 5 million cells per ml in the mould, enough to cover the knee cap. Cover the top using a filter paper soaked in 100 mMol/L CaCh

10) Wait 20 minutes to allow the ions to diffuse into the casted HATG, form an initial shell, and separate the casted knee cap from the mould

11) A cell-laden knee cap shaped scaffold with high precision to the original model is created.

1) Generate a 3D model of a mandibula from a CT/MRI scan

2) Print the 3D model using a DLP printer and a UV curable resin

3) Prepare an aqueous solution of 2.5 w/v % alginate with 100 mMol/L CaC0 ₃ and 100 mMol/L glucono delta-lactone

4) Pour the alginate solution on the 3D model and wait for the solution to crosslink

5) Remove the 3D model from the mould

6) Cast 0.5 w/v % HATG solution containing 1 million cells per ml

7) Place the mould with the casted solution in a sterile container and add cell culture media

8) Incubate at 37°C and 5% CO2 for 21 days

9) Remove the casted mandibula from the mould

1) From a CT/MRI scan of a patient requiring hip resurfacing

2) Print the 3D model of the hip allograft minus the cartilage layer using a DLP printer and a UV curable resin

3) Prepare an aqueous solution of 1 w/v % low melting agarose and 100 units per ml of tyrosinase

4) Pour the alginate solution on the 3D model and wait for the solution to crosslink 5) Remove the 3D model from the mould

6) Place allograft bone in the holder and cast 1.5 w/v % hyaluronic acid-tyramine solution containing 2 million cells per ml to add the cartilage layer.

7) Place the mould with the casted solution in a sterile container and add cell culture media

8) Incubate at 37°C and 5% CO2 for 21 days

9) Remove the casted graft from the mould

1) 3D-print an ear model using a DLP printer;

2) prepare an aqueous solution of kappa carrageenan 3 w/v %, locust bean gum 1 w/v % and 100 mMol/L CaCh and heat the solution to 80°C until complete solution of the polymer;

3) place and secure the 3D printed ear model inside a container with width and height at least as large as the ear model with a minimum of 10 mm of clearance on each side;

4) pour the kappa carrageenan and locust bean gum solution in the mould and leave to cool down to room temperature; prepare an additional sheet of a few centimetres of the same material in an identical container;

5) remove the ear model from the gelled mould (thereby creating an CaCh eluting mould); remove the sheet from the mould (thereby creating an CaCh eluting cap);

6) pour 1 % alginate solution in the mould;

7) wait 1 minute to allow CaCh ions to diffuse into the casted alginate, crosslinking a thin alginate shell;

8) remove the uncrosslinked alginate using a syringe;

9) pour 3% hyaluronic acid solution containing 10 million chondrocytes per ml, leaving a few millimetres at the top of the mould;

10) close the mould by pouring a 1 % alginate solution on top of the Hyaluronic Acid and closing the mould with the additional kappa carrageenan sheet created earlier;

11 ) wait 30 minutes and remove the casted ear from the mould;

12) a cell-laden ear shaped protective sleeve containing an un-crosslinked hydrogel is created.

Example 7: Lonp term stability of constructs cultured in alpinate shells , casted usinp an elutinp mold

Custom-shaped alginate shells were generated to be used as ‘culture chambers’. An eppendorf shaped 3D object was placed in a container and filled a 100 mM calcium-loaded, k-carragenan solution and left to cool down to room temperature to form a mold. The 3D object was then removed leaving an impress in the mold. Next, an alginate shell was formed by pouring a 1% alginate solution into the mold (Figure 4A). As ions started to elute from the mold into the alginate, they started forming a thin crosslinked alginate shell at the interface with the mold (Figure 4B). Once the desired thickness of 100 pm was reached, non- crosslinked alginate was aspirated (Figure 4C) leaving an alginate shell in the shape of the initial 3D printed object on the walls of the mold. The opening located at the top of the mold allows to fill the formed alginate shell with another infill material (Figure 4D,E). To seal the top part of the construct, additional alginate solution was poured on the infill material (Figure 4F) and finally crosslinked by placing another layer of eluting mold on top (Figure 4G). The top layer bonded with the already present surrounding shell (Figure 4H).

The thickness of the alginate shell can be tuned by changing the time at which the alginate polymer is subjected to the ion influx from the surrounding mold. The thickness of the shell can also be predicted via FEA analysis (Figure 6). The predictions correlate with the experimental results (Figure 7).

10 million cells per ml were embedded in the polymers of Table 1 and were cultured in vitro for 3 weeks. Tissue maturation of these materials was evaluated mechanically (Figure 8) and histologically (Figure 9). In fluid form materials, cells formed ‘condensed structures’ (Figure 10, 1% HA and 2% HA). In the weekly crosslinked and stabilized granular materials, cells remained spread out (Figure 10). Cell condensates matured their surrounding polymer reaching a 212.9±76.6 kPa Hertz modulus. For the other materials, the Hertz modulus remained homogeneous at around 100 kPa. Glycosaminoglycan (Safranin O) and collagen (I and II) deposition was observed in all conditions (Figure 9).

Table 1: Infill materials used

ID Type Polymer Cone. Description

1% HA Fluid Hyaluronan 1% Uncrosslinked hyaluronan 2% HA Fluid Hyaluronan 2% solubilized in PBS

Pure

Cells Fluid Dense chondrocyte solution

HA-MA Granular Hyaluronan 2% HA-MA microgels

Glo20 Granular Hyaluronan 2% Commercial facial filler based on

Glo30 Granular Hyaluronan 3% hydrogel particles

Alg-S Hydrogel Alginate-Sulfate 0.5% Crosslinked for 30 min by CaCh

Alg Hydrogel Alginate 1% diffusion from the mold

Example 8: Long term stability of constructs casted using an eluting mold

Human articular chondrocytes were embedded in a 1.5% HA-TG, 0.3% Alginate (HATG-AIg) hydrogel at a concentration of 10 million cells per ml. The crosslinking of both HA-TG and Alginate is triggered by calcium ions. 100 mM CaCI2 was preloaded in an 3% Agarose eluting mold loaded with 100 mM CaCI2 ions used to cast ear-shaped constructs (Figure 11). The shape fidelity after 3 weeks of culture of the casted polymer can be observed in Figure 11. Table 2 reports the deviation in size of the casted samples from the original ear. On average, there is only a mean deviation of 0.76% in the length, a 2.7% deviation in their width and a 3.1% deviation in the area between the casted and original DLP printed plastic ear used to generate the mold.

Tissue maturation of these materials was evaluated mechanically (Figure 12) and histologically. (Figure 13). The mechanical characterization of the casted constructs was performed via indentation testing using a Bioindenter (Antoon Paar). Several different locations were probed on each ear (Figure 12). The mean Hertz Modulus reported was of 5.8 kPa. Glycosaminoglycan (Safranin O) and collagen (I and II) deposition was observed in all conditions reaching similar intensity values as auricular cartilage, although a higher collagen I deposition was observed (Figure 13).

To conclude, the eluting mould system allows to fabricate patient specific grafts with high accuracy and reproducibility, effectively removing some of the main challenges associated with common casting techniques and bioprinting. This approach is universal and can be used with any pourable polymer. Furthermore, it allows to reduce the time required for crosslinking up to 80% compared to other casting techniques, while allowing cells to be embedded in large structures with minimal cell death.

Table 2: Shape stability of an 1.2% HA-TG, 0.3% Alginate ear casted using an Eluting Mold

Example 9: Modification of the eluting molds into multi-part molds with cuts defined with respect to the geometric and topological constraints involved in the process of the casting

In the current description of the eluting mold invention, the molding material is poured on top of the object to be cased, which can then be removed due to the flexible properties of the molding material. Nevertheless, some complex shape structures may now allow the object to be removed without rupturing the mold in some locations. For this reason, we have updated the eluting mold design into a multi-part eluting mold. This allows the same advantages as the eluting mold casting with the additional benefit of being able to separate and rejoin the different parts of the mold to extract the pattern object and casted hydrogel. Additionally, instead of simply cutting the molds planarly, the cuts are performed by following the geometrical constraints of the object that needs to be casted: Figure 5 shows the 3D design of the Eluting Molds that allow to cast an ear cartilage model. To generate these parts using the casting (eluting) material, the inverse of these models is modelled and printed using an SLA/DLP printer.

Example 10: Crosslinking behavior using eluting moulds

Figure 14 shows the increase in storage and loss modulus of 1% Alginate and 0.5% Alginate when crosslinked using a 100 mM CaCh loaded, 3% k-carragenan eluting mould as well as the storage and loss modulus of 1% Gelatin-tyramin and HA-tyramin when crosslinked using a 0.1% H2O2 loaded, 3% k-carragenan eluting mould. All materials crosslink fully within two hours reaching stiffnesses ranging in the kPa range.

Methods

Eluting mould preparation

Eluting mould solution was prepared by mixing 100 mM CaCI2 (Sigma) in mQ water with a 3% (w/v) k-carragenan solution ( GP Kelco). The solution was heated up to 95°C for 10 minutes before being poured. Once poured, the solution was left to cool down to room temperature.

Alginate and Gelatin Tyramin modification

Alginate's and Gelatin’s carboxylic groups were activated by aqueous carbodiimide chemistry resulting in the conjugation of N-hydroxysuccinimide (NHS; Sigma). Alginate/Gelatin-NHS esters were then coupled with tyramine hydrochloride (Sigma) for 24 hours at room temperature.

Alginate Sulfate

2% (w/v) UP-LVG alginate (Mw = 140 kDa, FG = 0.67) was dissolved in formamide, followed by addition of 96 % chlorosulfonic acid. The reaction was run at 60 °C for 2.5 hours. Alginate was then precipitated in cold acetone and centrifuged. Alginate was dissolved in mQ water by gradual neutralization using 5 M NaOH and dialyzed against 100 mM NaCI for 24 hours, followed by a second dialysis in mQ water for another 24 hours before being lyophilized.

Mechanical characterisation

Mechanical characterisation was performed using a 500 pm spherical ruby indentation probe on a Bioindenter (UNHT ³ Bio, Antoon Paar). Samples were cut in two. One half of the samples was used for indentation while the other half for histological analysis. Samples were fixed on a 35 mm diameter petri dish and submerged in 0.9% NaCI for indentation. Three different locations were probed for indentation: center, middle and edge region of the sample (as shown in Figure 3). Each region was probed three times by indenting the samples 75 pm. Data was analyzed in the Anton Paar’s software and plotted in Graphpad Prism (v 7.04).

Histology

Samples were fixed in 4% paraformaldehyde for 2 hours at room temperature after being washed four times with 0.9% NaCI and dehydrated by transfer to 20, 40, 60 and 70% of ethanol for 1 hour each. Samples were then embedded in paraffin (LogosJ, Milestone) and into 5 pm thick sections using a microtome. Tissue sections were adhered to polylysine slides (Polysine, Thermo Scientific) and stained with Safranin O according to standard protocols. For collagen type I and II, colorimetric, immunohistochemical stainings were performed. The two primary antibody types used were: mouse anti-collagen type I and goat anti-collagen type II. On the next day, samples were washed in PBS and remaining peroxidase activity was quenched with 0.3% H2O2 to prevent non-specific signals. After an additional washing step, secondary antibodies were added: goat, anti-mouse IgG (HRP, Abeam #6789) and goat, anti-rabbit IgG for collagen type I and type II respectively. Samples were incubated for 1 hour and washed in PBS, before DAB substrate (ab64238, Abeam) was added and left to react for precisely 3 minutes. Sections were washed and counterstained with Mayer’s hematoxylin solution. To preserve samples, slides were cover slipped with resinous mounting media (Eukritt) and imaged with a with a histology slide scanner (Pannoramic 250, 3D Histech).

Rheology

Rheological properties of different polymers were measured using an Anton Paar MCT 301 rheometer equipped with a 10 mm parallel plate geometry at 25 °C in a humid atmosphere with a gap distance of 0.5 mm. Rheological properties of samples were examined by oscillatory shear sweeps (1% strain, 1 Hz) to evaluate storage and loss modulus.