3D fat tissue structure, produced using light-based structuring curing, for use as a breast implant

Abstract

The invention relates to a 3D fat tissue structure for use as a breast implant for surgical, therapeutic, prophylactic or aesthetic reconstruction, construction or replacement of human breast tissue, wherein the 3D fat tissue structure has a matrix that is made of a biocompatible polymer and colonised with a plurality of biological cell types, wherein the colonisation with the plurality of cell types takes place during the construction of the matrix made of a biocompatible polymer, in the course of which a blood vessel structure network is formed within the 3D fat tissue structure, characterised in that, for the construction of the matrix made of a biocompatible polymer, light-based, structuring curing of a photopolymerisable or photocrosslinkable fluid is used. The invention also relates to the use of a 3D fat tissue structure as a breast implant in humans, wherein the 3D fat tissue structure has a matrix made of a biocompatible polymer, which is colonised with a plurality of cell types, wherein the colonisation with the plurality of cell types takes place during the construction of the matrix made of a biocompatible polymer, in the course of which a blood vessel structure network is formed within the 3D fat tissue structure, characterised in that, for the construction of the matrix made of a biocompatible polymer, light-based, structuring curing of a photopolymerisable or photocrosslinkable fluid is used.

Description

[0001] 3D adipose tissue scaffold produced by light-based structuring curing for use as a breast implant

[0002] The present invention relates to a 3D fatty tissue scaffold for use as a breast implant for the surgical, therapeutic, prophylactic, or aesthetic reconstruction, reconstruction, or replacement of human breast tissue. This scaffold is produced by light-based, structuring curing of a photopolymerizable or photocrosslinkable liquid to form a biocompatible polymer matrix. Furthermore, the present invention also relates to the use of a 3D fatty tissue scaffold as a breast implant (e.g., in the field of aesthetic breast modification / enlargement) produced by light-based, structuring curing of a photopolymerizable or photocrosslinkable liquid to form a biocompatible polymer matrix.

[0003] With approximately 2.3 million cases worldwide in 2020, breast cancer is the most commonly diagnosed cancer and affects one in eight women in their lifetime. Nevertheless, mortality rates in Germany have been steadily declining since the late 1990s in the 40-70 age group, and the 10-year survival rate after initial diagnosis was over 80% in 2018. One of the standard therapies is the complete surgical removal of the mammary gland (mastectomy), whereby in many cases the skin and nipple can be preserved, thus allowing for more natural forms of the

[0004] Breast reconstruction can be made possible. Conventional heterologous

[0005] Breast reconstruction relies on saline or silicone-filled implants, which, however, are not a lifelong solution and carry high risks of complications such as infections, capsular fibrosis, dislocations, pain, and functional limitations in the shoulder / arm area. As an alternative to implants, it is possible to reconstruct the removed breast using an autologous flap, in which tissue from another body region is transferred to the chest wall and adapted there. However, this procedure is significantly more complex and time-consuming, requires specific training for the surgeon, and carries risks at the donor site, such as the risk of muscle weakness, the formation of hernias, and noticeable scars in exposed body regions, as well as risks related to the transplant, such as the death of the body's own tissue.To overcome the limitations of conventional reconstruction methods, the ideal solution is an ideally complete, biological reconstruction with a living implant.

[0006] Of the numerous techniques in tissue engineering, the combination of 3D printing technology with biological materials and cells, known as bioprinting, is the most promising method for generating such complex tissue composites. Autologous tissue is harvested from the patient and, in conjunction with state-of-the-art biofabrication technology, used for breast reconstruction without causing the discomfort at the donor site typical of flap surgery. Alternatively, allogeneic material can also be used.

[0007] Compared to other organs, restoring the original function of the glandular tissue can be neglected in breast reconstruction if necessary. Quality of life, restoration of integrity, aesthetics, and haptic quality are crucial for successful treatment. Current research therefore focuses on generating scale-appropriate, vital, shape- and volume-stable adipose tissue that does not cause fibrotic capsule formation and can be assimilated by the body.

[0008] A further development of traditional saline or silicone-filled breast implants, which have no biological activity whatsoever, are hybrid approaches in which synthetic and biological materials are combined in the implant. For example, there are numerous studies in which a shape-giving and -retaining 3D support scaffold made of biodegradable polymers, such as polycaprolactone, is augmented with the patient's liposuction after implantation. The disadvantage of this method lies in the support material used, which, although biocompatible, takes up to three years to be completely broken down by the body. It is also significantly stiffer than natural fatty tissue and is therefore perceived as a foreign body. As a foreign material, perioperative complications such as inflammation and seromas are increasingly to be expected.Due to the disadvantages of hybrid breast adipose tissue reconstruction, recent research in this field is pursuing approaches that rely on alternative materials. Hydrogels offer the greatest potential, as they closely mimic the extracellular matrix of tissues. Living cells can be deposited directly onto these matrices and behave similarly to native tissues. To replicate the complexity of anatomical structures, it is necessary to precisely control the spatial arrangement of cells. Modern 3D printing processes enable this indirectly through the controlled deposition of biomaterial along a pre-designed construct architecture (production of a hydrogel scaffold) followed by seeding with living cells. This has so far been achieved, for example, by using recombinant type I collagen from genetically modified tobacco plants in extrusion-based 3D printers.The resulting 3D-printed hydrogel scaffolds made of collagen and synthetic polymers are then seeded with cells. However, due to the excellent biocompatibility of hydrogels, it would be desirable to directly print living cellular material into a desired shape, making a subsequent seeding step obsolete.

[0009] However, this approach has not yet been successfully implemented. The direct combination of autologous fat with biomaterials is intended to enable efficient healing of the bioimplant by the body and the formation of autologous tissue. This would result in a rapid and permanent improvement in quality of life after mastectomy, ideally simplifying future breast examinations and making subsequent reconstructive surgeries obsolete.

[0010] Regenerative medicine deals with the treatment of various diseases through the restoration of dysfunctional cells, tissues, and organs. Tissue engineering focuses on biological replacement, for example, using cultured tissue. In recent years, the fields of application of regenerative medicine have increasingly expanded to include tissue replacement, whereas originally this area was primarily comprised of stem cell therapies, for example, for the treatment of leukemia.

[0011] The structure of these three-dimensional tissues and cell networks is becoming increasingly complex. Cellular self-organization can no longer ensure the construction of these complex structures. Therefore, the invention relies on technical aids such as 3D printing, or more precisely, "bioprinting." Bioprinting allows the positioning, shaping, and production of complex constructs using 3D printing with cells and biopolymers. However, due to the complexity of these processes, the state of the art has not succeeded in producing a corresponding tissue that can be successfully produced in vitro for subsequent implantation into the human body.

[0012] It is therefore the object of the present invention to produce a 3D adipose tissue scaffold (3D scaffold) for use as a breast implant that overcomes the aforementioned disadvantages. In particular, the object of the present invention is to provide a 3D adipose tissue scaffold that closely resembles natural breast adipose tissue. According to the invention, it is desirable for the 3D scaffold to have a tissue structure, both geometrically and in terms of material, in which dedicated compartments consisting of, on the one hand, adipose tissue and, on the other hand, vascular structural tissue with a continuous blood vessel network are present.

[0013] For this purpose, according to the invention, a 3D fatty tissue scaffold is provided for use as a breast implant for the surgical, therapeutic or prophylactic reconstruction, construction or replacement of human breast tissue, wherein the 3D fatty tissue scaffold has a matrix made of a biocompatible polymer which is populated with a plurality of biological cell types, wherein the colonization with the plurality of cell types takes place during the construction of the matrix from biocompatible polymer with the formation of a blood vessel structure network within the 3D fatty tissue scaffold, characterized in that light-based, structuring curing of a photopolymerizable or photocrosslinkable liquid is used for the construction of the matrix from biocompatible polymer.

[0014] In other words, the present invention also relates to the use of a 3D fatty tissue scaffold for producing a breast implant for the surgical or therapeutic reconstruction, construction or replacement of human breast tissue, wherein the 3D fatty tissue scaffold has a matrix made of a biocompatible polymer which is populated with a plurality of biological cell types, wherein the colonization with the plurality of cell types takes place during the construction of the matrix from biocompatible polymer with the formation of a blood vessel structure network within the 3D fatty tissue scaffold, characterized in that light-based, structuring curing of a photopolymerizable or photocrosslinkable liquid is used for the construction of the matrix from biocompatible polymer.

[0015] Surgical or therapeutic reconstruction or replacement refers to the reconstruction or replacement of diseased breast tissue removed from a patient through the insertion of a breast implant. The (surgical, therapeutic, or prophylactic) reconstruction of human breast tissue also includes breast augmentation, i.e., the insertion of a breast implant in addition to the existing breast tissue. The latter can be performed for medical or aesthetic reasons.

[0016] In one embodiment of the 3D fatty tissue scaffold according to the invention, it is preferred that the human breast tissue has been removed as a result of a disease. This can involve the partial removal of fatty breast tissue, but also the complete removal of the fatty breast tissue including the mammary gland. It is particularly preferred that the disease is a tumor, in particular breast cancer.

[0017] The term “made of biocompatible polymer” preferably means that the 3D scaffold is constructed from a matrix of one or more biocompatible polymers.

[0018] By “a plurality of cell types” it is to be understood that the 3D scaffold according to the invention has different cell types that mimic different properties of healthy adipose tissue in a manner that is as body-like as possible.

[0019] The term "colonization with the majority of cell types during the construction of the matrix from biocompatible polymer" is to be understood here as meaning that the different cell types are not introduced after the construction of the matrix, i.e. the matrix is ​​not colonized, but the colonization with the different cell types takes place simultaneously with the light-based, structuring curing of the photopolymerizable or photocrosslinkable liquid to the matrix from biocompatible polymer.

[0020] The light-based, structuring curing of a photopolymerizable or photocrosslinkable liquid to form the biocompatible polymer can be any exposure method suitable for building a matrix of a biocompatible polymer. Here, a photopolymerizable or photocrosslinkable liquid is cured to a biocompatible polymer by the structuring irradiation of light (electromagnetic radiation). In particular, according to the invention, a stereolithographic 3D printing method is used for the light-based, structuring curing, particularly preferably one that is a projection-based exposure method, i.e. in which curing takes place in a focal plane, i.e. preferably non-point curing. Such a method is described in EP 3 018 531 A1 and is preferably used according to the invention.

[0021] For this purpose, it is preferred that the one or more photopolymerizable or photocrosslinkable liquids comprise at least one cell type and one photopolymerizable or photocrosslinkable substance.

[0022] In one embodiment of the 3D scaffold according to the invention, it is therefore preferred that its construction be carried out using a stereolithographic 3D printing process. A stereolithographic 3D printing process is understood to be one in which the structure of the 3D scaffold is gradually produced by point-by-point, layer-by-layer (in a single layer), or holographic curing, with curing within a single layer being preferred according to the invention.

[0023] In one embodiment of the 3D scaffold according to the invention, it is preferred that the matrix of the biocompatible polymer is constructed by the following step:

[0024] (i) Curing a photopolymerizable or photocrosslinkable liquid by irradiating electromagnetic radiation pointwise, in a layer or holographically, in the region of the photopolymerizable or photocrosslinkable liquid in which curing or the construction of the 3D framework is desired, wherein irradiation in a layer is preferred according to the invention.

[0025] Irradiation in a layer is to be understood here in particular as meaning that the 3D framework according to the invention to be built up is irradiated across its entire surface while it is in the photopolymerizable or photocrosslinkable liquid. In this case, only those areas of the surface where spatial curing is desired are irradiated. Areas of the surface where curing is not desired may also not be irradiated. In a further step (i), however, a region with a different structure than in the first step (i) can then also be produced in the same layer of the 3D framework according to the invention to be built up by introducing the layer into another photopolymerizable or photocrosslinkable liquid and correspondingly irradiating the desired area of ​​the layer.Furthermore, the 3D framework according to the invention can also be constructed by repeating step (i) and applying additional cured areas of the same or different structure and / or composition to the already cured areas. In this way, a complex 3D framework with different structures in each layer can be obtained.

[0026] For curing in one layer (projection-based or flat), the so-called “digital light processing” is preferably used.

[0027] Preferably, the photopolymerizable or photocrosslinkable substance is present in the photopolymerizable or photocrosslinkable liquid in liquid form, for example dissolved in a solvent.

[0028] In order for the matrix made of the biocompatible polymer to be populated with a plurality of cells, the photopolymerizable or photocrosslinkable liquid used contains one or more cell types.

[0029] Step (i) is preferably repeated by renewed irradiation of electromagnetic radiation selectively, in a layer, or holographically, with irradiation in a layer being preferred according to the invention. When repeating step (i), a further photopolymerizable or photocrosslinkable liquid is preferably used. The further photopolymerizable or photocrosslinkable liquid can be identical to or different from that used in the first step. The latter can be different either with regard to the photopolymerizable or photocrosslinkable substance, with regard to the concentration of individual components, or with regard to the cell type(s) used. The same applies to photopolymerizable or photocrosslinkable liquids used in further steps.

[0030] "Photopolymerizable" is understood herein to mean that the corresponding substance contains monomers that can be polymerized by exposure to electromagnetic radiation and, optionally, the presence of a photoinitiator. Likewise, "photocrosslinkable" is understood to mean that an oligomer or polymer can be crosslinked by exposure to electromagnetic radiation and, optionally, the presence of a photoinitiator.

[0031] In one embodiment of the 3D scaffold according to the invention, it is preferred that the matrix of the biocompatible polymer is constructed by the following steps (preferably in the order given):

[0032] (I) introducing a first photopolymerizable or photocrosslinkable liquid into a reaction vessel,

[0033] (II) immersion or presence of a support plate on which the 3D scaffold is to be constructed in the first photopolymerizable or photocrosslinkable liquid;

[0034] (III) Irradiation of electromagnetic radiation selectively, in a layer or holographically in the areas where curing of the liquid is desired

[0035] (IV) producing a polymerized or cross-linked structure by the electromagnetic radiation, (V) introducing another photopolymerizable or photocross-linkable liquid into a (further) reaction vessel,

[0036] (VI) immersion or presence of the structure produced in step (IV) on the carrier plate in the further photopolymerizable or photocrosslinkable liquid,

[0037] (VII) irradiating electromagnetic radiation selectively, in a layer or holographically in the areas where curing of the further liquid is desired, (VIII) producing a further polymerized or cross-linked structure by the electromagnetic radiation in step (VII),

[0038] (IX) Repeating steps (V) to (VIII) with another photopolymerizable or photocrosslinkable liquid until the 3D scaffold is produced.

[0039] The polymerized or cross-linked structures produced in the repeated steps are preferably linked together by covalent bonds. However, non-covalent bonds, for example, based on physical interactions, are also possible.

[0040] By repeating the process steps in which the photopolymerizable or photocrosslinkable liquids polymerize or crosslink, a layered, point-by-point, or holographic structure of the 3D scaffold is achieved. This makes it possible to construct a 3D scaffold with a complex structure, in which the various cell types used are located at different, desired positions within the 3D scaffold. Furthermore, undercuts and overhanging structures can also be formed, since polymerization or crosslinking of the photopolymerizable or photocrosslinkable liquid can occur in a specific layer or specific point during point irradiation, even if there is no already polymerized or crosslinked material underneath, but merely an unpolymerized or uncrosslinked liquid.In this way, the invention also makes it possible to construct a blood vessel structure network. Polymerization or crosslinking of a photopolymerizable or photocrosslinkable liquid located outside the layer or dot does not occur; rather, only the photopolymerizable or photocrosslinkable liquid located within the layer or dot is polymerized or crosslinked.

[0041] The photopolymerizable or photocrosslinkable liquids used preferably each contain one or more biological cell types. If polymerization or crosslinking occurs as a result of exposure to electromagnetic radiation, the cells contained in the liquid are embedded in a corresponding polymer. By using several photopolymerizable or photocrosslinkable liquids, preferably with one or more different biological cell types, a complex 3D scaffold can be constructed in the form of a biological breast implant, which also has a blood vessel structure network. The use of a matrix made of the biocompatible polymer enables the directed construction of the 3D scaffold according to the invention.

[0042] The matrix of the biocompatible polymer can be composed of a homogeneous material and thus comprise only a polymer of a single type, but can also be composed of a heterogeneous material made up of biopolymers of different types, whereby only the matrix-forming material without the cell types embedded therein is meant.

[0043] A biocompatible polymer is understood to be a biological or biocompatible polymer. "Biocompatible" is understood to mean that it does not affect the lifespan of biological cells, in particular, that it does not have a toxic effect on them. Furthermore, it is preferred that the biocompatible polymer is a biocompatible hydrogel. A hydrogel is a gel made of a water-insoluble polymer that can bind water. Furthermore, it is preferred that the biocompatible polymer is a biodegradable or digestible polymer. This allows the polymer to be gradually broken down or digested after implantation into the human body, leaving only the autologous cells as the implant.The photopolymerizable or photocrosslinkable substance in the photopolymerizable or photocrosslinkable liquid is preferably one that has a photoreactive group that can form covalent bonds with other photoreactive groups.

[0044] In one variant, the photoreactive group is an acrylic group, by means of which the polymerization or crosslinking is achieved. This means that the photopolymerizable or photocrosslinkable substance is preferably one of the following group: methacrylic acid, methacrylates, methyl acrylates, ethyl acrylates, hydroxyethyl acrylates, butyl acrylates, trimethylolpropane acrylates, triacrylacrylates, and polyacrylates (PA) in general.

[0045] The substance to be polymerized or crosslinked can be a polymer, an oligomer, or a monomer. These are preferably carbon-based substances. Monomers are photopolymerized. Polymers or oligomers are preferably photocrosslinked.

[0046] The following monomers can be used, for example, as polymerizable monomers: acrylamides, vinyl chloride, ethylene, propylene, isoprene, caprolactam, caprolactone lactide, all amino acids, (de-)oxyribonucleotides, glucose, or all monosaccharides as well as the aforementioned acrylates, with caprolactone, lactide, monosaccharides, (de-)oxyribonucleotides being preferred for reasons of biodegradability of the resulting polymers.

[0047] The following can be used as oligomers or polymers: polycaprolactone, polylactide, polyethylene glycol (PEG), polyethylene (PE), polypropylene (PP), polyketone (PK), polyvinyl chloride (PVC), polystyrene (PS), polytetrafluoroethylene (PTFE), polymethyl methacrylate (PMMA), polycarbonate (PC), polyethylene terephthalate (PET), and polyurethane (PU). Synthetic polymers such as silicones, polydimethylsiloxane (PDMS), or resins such as melamine or melamine-formaldehyde resins are also suitable as starting materials. Biopolymers such as proteins, DNA, RNA, carbohydrates and carbohydrate derivatives, collagens, fibrins, alginates, gelatin, hyaluronic acids, or polylactides are also suitable as starting materials. Instead of the aforementioned polymers, the monomer precursors or oligomer precursors of these polymers can also be used as starting materials, provided they can be provided in a stable manner in the solid or liquid state.By incorporating a photoreactive group, such as an acrylic group, into the starting substance, it is rendered photopolymerizable or photocrosslinkable. The radiation-induced coupling of the acrylic residues between different molecules of the starting substance produces a polymerized or crosslinked matrix. Polycaprolactone, polylactides, and all biological polymers are particularly preferred for reasons of biodegradability.

[0048] The starting substance, supplemented with the photoreactive group, is used in liquid form, with different viscosities possible. This means that the process described here is not limited to photopolymerizable or photocrosslinkable liquids with a specific viscosity; low-viscosity liquids can also be used. Both Newtonian and non-Newtonian liquids can be used.

[0049] The liquids can be solutions or colloidally dispersed mixtures, such as suspensions. The liquids can range in character from aqueous to oily. This is determined, among other things, by the choice of starting substances and their particle size.

[0050] In order to achieve photopolymerization or photocrosslinking of the starting substance carrying a photoreactive group, a radical former (a so-called photoinitiator) is preferably used, which forms radicals at a selected wavelength of the electromagnetic radiation used in the process. Suitable radical formers are, for example, anthrone derivatives such as violanthrone or isoviolanthrone, fluorescein, rubrene, anthrazine derivatives, tetrazene derivatives, benzanthrone, benzanthronil, eosin, levolinic acid derivatives, phosphine derivatives, mono- and bis-acyl phosphines, in particular lithium phenyl-2,4,6-trimethylbenzoylphosphinate, metallocenes, acetophenones, benzophenones, xanthones, quinones, ketone derivatives, hydroxyketones, aminoketones, benzoyl peroxides, pyridine salts, phenylglyoxylates and / or iodonium salts.In addition to the radical generator, a vinyl macromer and an amine-based co-initiator are preferably used to ensure that the photopolymerization or photocrosslinking proceeds in a particularly suitable manner. Suitable co-initiators include, for example, ascorbic acid and tertiary amine derivatives such as methyldiethanolamine or tetraethylamine.

[0051] In addition, a substance can be added to the photopolymerizable or photocrosslinkable liquid to prevent photopolymerization or photocrosslinking of deeper, liquid layers. This ensures that a liquid solution outside the focal plane remains liquid, even if it is within the incident beam of the focal plane above it. This works through absorption of the substance at the wavelength at which polymerization occurs (polymerizing wavelength). The absorption takes place in the focal plane, preventing the polymerizing wavelength from penetrating deeper layers. Suitable substances include all substances that absorb at the desired wavelength, such as dyes.

[0052] Furthermore, in one variant, it is possible for the one photopolymerizable or photocrosslinkable liquid and / or one of the other photopolymerizable or photocrosslinkable liquids and / or another liquid that does not have to be photopolymerizable to contain a temperature-sensitive gelling agent. In particular, the use of an inverse temperature-sensitive (also referred to as reverse temperature-sensitive) gelling agent is envisaged. Such a gelling agent becomes more solid with increasing temperature. By heating the reaction vessel, the reaction liquid solidifies and initially forms a metastable gel. If the liquid is not photopolymerized or photocrosslinked at the same time, the metastable gel can be liquefied again and pumped out by subsequent cooling of the 3D scaffold. With conventional temperature-sensitive gelling agents, the temperature conditions to be used are exactly the opposite.For example, a support structure can be created if necessary, allowing the creation of hanging structures. However, if the metastable gel is at least partially irradiated with electromagnetic radiation of a suitable wavelength, photopolymerization occurs, transforming the metastable gel in these areas into a stable gel or polymer. In other words, the temperature-sensitive, particularly inverse-temperature-sensitive, gel former and temperature-controlled reaction chamber make it even easier to work with hanging sections, undercuts, or cavities. Even in this variant, liquid structures can still be used as supports.

[0053] It is also possible to provide a temperature gradient so that a metastable gel does not form in all regions of the liquid containing the temperature-sensitive, particularly inverse-temperature-sensitive, gelling agent. Using such a gradient, even more complex structures can be created.

[0054] The aforementioned individual components can be contained as individual substances in the photopolymerizable or photocrosslinkable liquid. Alternatively, it is also possible to realize the substances or groups preferably used for gel formation in a single polymer by appropriate synthesis. Instead of a mixture of individual components, such a polymer would then have different functional groups that combine all the functions required or preferably used for photopolymerization or photocrosslinking. Furthermore, it is also conceivable to provide only some of the functions or groups preferably used for photopolymerization or photocrosslinking in one polymer and to mix other functions or groups preferably used for photopolymerization or photocrosslinking in separate individual components of the photopolymerizable or photocrosslinkable liquid.

[0055] Alternatively or in addition to the creation of cavities using a gelling agent, enzymes can also be used to digest the polymer. The principle is as follows: A 3D scaffold with cavities / undercuts (e.g., a channel system) is printed as a solid body. All cavities are filled with a sacrificial material during printing, which can later (i.e., after printing is complete) be dissolved by adding the appropriate enzyme. The sacrificial material is, for example, a digestible polymer that is digested by adding a digesting enzyme. This is an elegant strategy for creating cavities using stereolithographic printing processes. For example, a hyaluronidase (digesting enzyme) can digest hyaluronic acid (sacrificial material), creating a cavity at the location in the 3D scaffold where the hyaluronidase is inserted. This principle is already described in the patent application with the official file number DE 102019 200 792.9.

[0056] Alternatively, a photoblocker can be used in the photopolymerizable or photocrosslinkable liquid to create a cavity / undercut. The photoblocker limits the curing depth of the photopolymerizable or photocrosslinkable liquid. This allows for single-layer curing by irradiation without curing all areas within the beam path. The option of layer-by-layer curing increases printing speed immensely compared to spot-based processes.

[0057] In one variant, the additional photopolymerizable or photocrosslinkable liquid is preferably only introduced into a reaction vessel once the photopolymerizable or photocrosslinkable liquid previously present in the reaction vessel (this can be, for example, one photopolymerizable or photocrosslinkable liquid or another photopolymerizable or photocrosslinkable liquid) has been removed from the reaction vessel. For this purpose, it is possible, for example, to provide a pump that pumps a previously used photopolymerizable or photocrosslinkable liquid out of the reaction vessel and pumps a new, additional photopolymerizable or photocrosslinkable liquid into the reaction vessel. Instead of a single pump, two or more different pumps can also be used for such processes.Alternatively, the entire 3D scaffold to be constructed can be built on a carrier plate, which can be moved in such a way that the 3D scaffold is moved one after the other into different reaction vessels filled with photopolymerizable or photocrosslinkable liquid, where the light-based, structuring curing is carried out, as described above.

[0058] In one variant, an optical system is arranged between a source of electromagnetic radiation (radiation source), which serves to generate one and / or the further electromagnetic radiation, and the reaction vessel. This optical system serves to focus the electromagnetic radiation onto the respective focal plane in the reaction vessel. In one variant, it is provided that this optical system can be refocused in order to change the curing layer within the reaction vessel. Such refocusing can be achieved, for example, by changing the distance of the optical system from the radiation source. A computer-controlled stepper motor can be provided to mediate the corresponding movement of the optical system.The optical system can, for example, be a system of optical lenses or—in a particularly simple case—a single focusing lens. Alternatively, the curing point or layer can be static, and the 3D framework to be built can be moved relative to the electromagnetic radiation.

[0059] In a further manufacturing variant of the 3D scaffold according to the invention, the one and / or the further electromagnetic radiation is directed onto a defined and predeterminable area located within the one photopolymerizable or photocrosslinkable liquid and / or the further photopolymerizable or photocrosslinkable liquid. This means that a specific radiation pattern can be specified that impinges on the photopolymerizable or photocrosslinkable liquid and serves to polymerize or crosslink the liquid at these locations to form the biocompatible polymer. Such a radiation pattern can be generated, for example, by using masks or apertures, but also by using pulsed radiation or the digital modulation of a radiation signal.Polymerization or crosslinking occurs in the areas of the photopolymerizable or photocrosslinkable liquid that are exposed to the radiation. In the other areas not exposed to the radiation, the photopolymerizable or photocrosslinkable liquid remains in its unpolymerized or uncrosslinked state. The radiation thus defines the areas where printing of the polymerized or crosslinked structure takes place. With this type of light-assisted printing, much higher resolutions are possible than is the case with prior art processes. The resolution depends on the wavelength of the radiation used. Even with regularly used long wavelengths, it is better than the resolution achievable with conventional prior art processes.The more precisely the radiation source can be focused, the greater the resulting resolution. For example, very high resolutions can be achieved with a laser.

[0060] If necessary, the electromagnetic radiation can be directed to the respective layer or point using mirrors.

[0061] The selected irradiation pattern can, for example, be provided by a computer program. For example, it is conceivable for a user to create the 3D scaffold to be manufactured using a CAD program. The digital object created in this way is then cut into individual irradiation planes by a suitable computer program. Furthermore, a specific photopolymerizable or photocrosslinkable liquid is assigned to each plane, or to different locations within each plane. Control information for a printer, which is used to carry out the described process, is created from this information. This control information specifies when which photopolymerizable or photocrosslinkable liquid must be introduced into the reaction vessel. Furthermore, this control information specifies when which image of an irradiation plane should be projected onto the respective focal plane in the reaction vessel.In this way, the 3D framework previously created on the computer can be converted into a real 3D framework.

[0062] In one variant, more than one polymerized or cross-linked structure is created in the same layer. This involves first polymerizing or cross-linking a first photopolymerizable or photocross-linkable liquid. The 3D scaffold to be constructed is then introduced into a second photopolymerizable or photocross-linkable liquid. Preferably, those areas of the 3D scaffold to be constructed that were not previously irradiated and therefore do not yet contain a polymerized or cross-linked structure are then irradiated. This allows different materials and cell types to be arranged in one and the same layer or in layers on top of one another, depending on how far the 3D scaffold to be constructed is introduced into the second liquid. Consequently, several polymerized or cross-linked structures comprising different materials and cell types are formed in one and the same layer or the 3D scaffold.In this way, even complex structures, such as blood vessel networks, can be incorporated into the 3D scaffold. The 3D scaffold to be constructed can then be placed in another photopolymerizable or photocrosslinkable liquid containing another cell type, and so on. This makes it possible to place appropriate materials and cell types anywhere within the 3D scaffold, enabling the construction of a complex biological breast implant that closely resembles human body tissue.

[0063] In one variant, the first electromagnetic radiation and / or the further electromagnetic radiation have a wavelength in the range of 200 nm to 1000 nm (i.e., a wavelength lying between the UV and infrared ranges), more preferably in the range of 350 nm and 800 nm. Such wavelengths are particularly effective at exciting the substances preferably used as radical generators, so that radicals are formed to enable polymerization or crosslinking of starting substances containing acrylic residues.

[0064] Other suitable wavelengths of the electromagnetic radiation used are in the range from 250 nm to 950 nm, in particular from 250 nm to 850 nm, in particular from 300 nm to 800 nm, in particular from 300 nm to 750 nm, in particular from 300 nm to 700 nm, in particular from 350 nm to 650 nm and very particularly from 350 nm to 400 nm.

[0065] The radiation used for polymerization or crosslinking can comprise the same wavelength or different wavelengths from the aforementioned wavelength range to enable suitable polymerization of the different photopolymerizable or photocrosslinkable liquids. The individual radiations can be generated by different or by the same radiation source. It is also possible to use different wavelengths consecutively within a layer (and thus within a focal plane) to polymerize or crosslink different photopolymerizable or photocrosslinkable liquids in the same layer if a heterogeneous layer composed of different polymerized or crosslinked structures is to be formed.

[0066] As can be seen from the previous description of the production of the 3D scaffold according to the invention, the step of 3D printing the 3D scaffold can be carried out completely automatically, so that user intervention is not required. This further facilitates the application of the method.

[0067] The duration of the electromagnetic radiation applied to the respective focal plane can be adapted to the specific requirements of the photopolymerizable or photocrosslinkable liquids used. This means that each material is allowed a curing time that is necessary and appropriate for the desired polymerization or crosslinking.

[0068] If the 3D scaffold is created on a support plate, this support plate can be completely lifted out of the remaining liquid in the reaction vessel after the manufacturing process. The generated 3D scaffold can then be removed from the support plate by the user.

[0069] In one embodiment of the 3D fatty tissue scaffold according to the invention, it is preferred that human breast tissue has been partially or completely removed (mastectomy) for the prevention of or as a result of a disease. The disease is particularly preferably breast cancer. The removal of breast tissue for the prevention of a disease preferably results from the determination of a genetic predisposition that, for example, increases the risk of developing cancer, preferably a genetic predisposition to breast cancer. In a further embodiment of the 3D fatty tissue scaffold according to the invention, it is preferred that the cells of the majority of cell types are autologous cells. Autologous cells are understood to be the body's own cells, i.e. the cells used to construct the 3D fatty tissue scaffold are those that were removed from the patient who is to receive the breast implant.In this way, rejection reactions of the implanted breast implant are unlikely to be expected.

[0070] The cells of the majority of cell types are preferably selected from the group consisting of adipocytes, adipocyte progenitor cells, adipocyte stem cells, fibroblasts, endothelial cells, and a combination thereof. It is preferred that adipocyte stem cells be used for the light-based, structuring curing, which then subsequently develop into adipocytes. To support the further development of adipocytes into adipocyte stem cells, cellular messenger substances can be used according to the invention. So-called adipogenic differentiation factors are preferably used for this purpose. These include signaling molecules from the classes of growth factors and hormones. The adipogenic factors are preferably added to the nutrient fluid for the culture of the 3D adipose tissue scaffold in which the adipocyte stem cells are located.

[0071] The aforementioned cell types are preferably introduced into one or more photopolymerizable or photocrosslinkable liquids. Each cell type can be introduced into a separate photopolymerizable or photocrosslinkable liquid. Alternatively, two (or more) cell types can be introduced into one photopolymerizable or photocrosslinkable liquid.

[0072] In one embodiment of the 3D adipose tissue scaffold according to the invention, it is preferred that a first photopolymerizable or photocrosslinkable liquid containing adipocyte stem cells and a second photopolymerizable or photocrosslinkable liquid containing endothelial cells or fibroblasts be used for the light-based, structuring curing. It is further preferred that the second photopolymerizable or photocrosslinkable liquid contain endothelial cells. In the latter case, fibroblasts can either be additionally contained in the first photopolymerizable or photocrosslinkable liquid, or a third photopolymerizable or photocrosslinkable liquid containing fibroblasts is additionally used for the light-based, structuring curing.

[0073] Preferably, however, the adipocyte stem cells and the fibroblasts are placed in a single photopolymerizable or photocrosslinkable fluid, and the endothelial cells are placed in another photopolymerizable or photocrosslinkable fluid. In this way, the cells that perform tissue function (adipocytes) can be placed at different locations within the 3D scaffold from the blood vessel formation cells during the construction of the 3D scaffold. This enables the construction of a blood vessel structural network.

[0074] The 3D adipose tissue scaffold according to the invention preferably comprises adipocytes and / or adipocyte stem / progenitor cells, and another cell type. The other cell type is preferably selected from fibroblasts and endothelial cells, with endothelial cells being preferred. More preferably, the 3D adipose tissue scaffold according to the invention comprises adipocytes and / or adipocyte stem / progenitor cells, fibroblasts, and endothelial cells. The adipocytes are responsible for fat / energy storage and the hormonal activity of the 3D scaffold. The fibroblasts in the 3D scaffold are responsible for the secretion of extracellular matrix and for supporting vascularization. The endothelial cells are responsible for blood vessel formation (vasculogenesis and angiogenesis) and tissue supply.

[0075] Furthermore, in the 3D adipose tissue scaffold according to the invention, it is preferred that the light-based, structuring curing be a stereolithographic 3D printing process. The stereolithographic 3D printing process is—as mentioned above—preferably a projection-based exposure process.

[0076] An exposure process involving patterned curing of a photopolymerizable or photocrosslinkable substance to form the biocompatible polymer—as used in the invention—has the advantage over other biological printing processes, such as the extrusion process, the inkjet process, and the laser-assisted forward transfer process, of superior printing speed, i.e., the volume of the object to be printed that can be built up per unit of time. Unique to light-based patterned curing is the combination of high printing speed and high spatial resolution. The process's performance is based on optical principles, and the use of commercially available optics enables superior resolution down to the submicrometer range.

[0077] In one embodiment of the breast implant according to the invention, it is preferred that diseased breast tissue is removed from the patient in one step. Furthermore, it is preferred that healthy adipocyte stem cells, fibroblasts, and endothelial cells are removed from the patient. The removed, healthy cells are then preferably cultured. By adding the cultured cells, the various photopolymerizable or photocrosslinkable liquids used for constructing the 3D scaffold according to the invention are then preferably produced—as described above. After constructing the 3D scaffold, the interior of the formed blood vessel structure network is preferably enzymatically digested to expose the channels. In one variant, the 3D scaffold is cultivated in a further intermediate step to form tissue structures.The implant can then be implanted into the patient.

[0078] The present invention relates not only to the 3D scaffold according to the invention, but also to the use of a 3D fatty tissue scaffold as a breast implant in humans, wherein the 3D fatty tissue scaffold comprises a matrix made of a biocompatible polymer populated with a plurality of cell types. The colonization with the plurality of cell types occurs during the construction of the matrix made of biocompatible polymer, forming a blood vessel structure network within the 3D fatty tissue scaffold. Light-based, structuring curing of a photopolymerizable or photocrosslinkable liquid is used to construct the matrix made of biocompatible polymer. This is preferably a non-medical indication.All definitions and process steps for the production of the 3D scaffold according to the invention mentioned above are also intended to apply to the 3D fatty tissue scaffold used in the use according to the invention.

[0079] Various results are shown in the figures, as they are also described in more detail in the examples section below:

[0080] Fig. 1a to 1c show the differentiation of hASCs into adipocytes

[0081] (Control group in 2D culture).

[0082] Fig. 2a and 2b show the histological and electron microscopic

[0083] Detection of formed fatty tissue.

[0084] Fig. 3a to 3c show the vascularization of the bioprinted adipose tissue.

[0085] Examples: Preparation of a photopolymerizable or photocrosslinkable liquid (without cells):

[0086] Porcine gelatin type A (300 Bloom, Sigma-Aldrich, USA) is methacrylated as described in [1] and [2]. Briefly, 10% w / v gelatin is dissolved in phosphate-buffered saline (PBS) and heated to 50 °C. Methacrylic anhydride (Sigma-Aldrich, USA) is added dropwise (0.1 mL / gelatin), and the reaction mixture is stirred for three hours to obtain methacrylated gelatin (GelMA). After the pH of the solution is neutralized, it is dialyzed against distilled water (12-14 kDa cut-off membrane) for four days to desalt and remove free methacrylate. It is then freeze-dried (Alpha 1-4 LDplus, Martin Christ, Germany, -60 °C, 1 mbar) to obtain a long-lasting lyophilizate.Hyaluronic acid (HA) from Streptococcus equi (molecular weight >1 MDa, Alfa Aesar, USA) is autoclaved to reduce the chain length, and then methacrylated hyaluronic acid (HAMA) is synthesized following a modified protocol of Poldervaart et al. [3], Briefly, hyaluronic acid is dissolved in ultrapure water and the solution is adjusted to pH 9.0. Then, 2.0 ml / gHA of methacrylic anhydride dissolved in dimethyl sulfoxide (VWR, UK) is added and stirred for 24 hours at room temperature. The product is purified by dialysis against ultrapure water and subsequently lyophilized. The photoinitiator lithium phenyl 2,4,6-trimethylbenzoylphosphinate (LAP) is synthesized as described elsewhere [4][5], The degree of functionalization of the synthesized GelMA and HAMA is determined by. 1 H-NMR using a Bruker Avance III at 500 MHz (Bruker Corporation, USA).

[0087] [1] a I. Van Den Bulcke, B. Bogdanov, N. De Rooze, E. H. Schacht, M.

[0088] Cornelissen, and H. Berghmans, “Structural and rheological properties of methacrylamide modified gelatin hydrogels.,” Biomacromolecules, vol. 1 , no. 1 , pp. 31-38, 2000, doi: 10.1021 / bm990017d.

[0089] [2] H. Shirahama, B. H. Lee, L. P. Tan, and N. J. Cho, “Precise tuning of facile one- pot gelatin methacryloyl (GelMA) synthesis,” Scientific Reports, vol. 6, no. August, pp. 1-11 , 2016, doi: 10.1038 / srep31036. [3] M. T. Poldervaart et al., “3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity,” PLoS ONE, vol. 12, no. 6, pp. 1- 15, 2017, doi: 10.1371 / journal.pone.0177628.

[0090] [4] T. Majima, W. Schnabel, and W. Weber, “Phenyl-2,4,6- trimethylbenzoylphosphinates as water-soluble photoinitiators. Generation and reactivity of ODP(C6H5)(O-) radical anions,” Die Makromolekulare Chemie, vol. 192, no. 10, pp. 2307-2315, Oct. 1991 , doi: 10.1002 / macp.1991.021921010.

[0091] [5] B. D. Fairbanks, M. P. Schwartz, C. N. Bowman, and K. S. Anseth, “Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6- trimethylbenzoylphosphinate: polymerization rate and cytocompatibility,” Biomaterials, vol. 30, no. 35, pp. 6702-6707, 2009, doi:

[0092] 10.1016 / j. biomaterials.2009.08.055.

[0093] Herstellung der Zellkulturen:

[0094] Various cell cultures are prepared. Human adipose stem cells (hASCs), dermal fibroblasts (hDFs), and umbilical vein endothelial cells (HllVECs) are purchased from Lonza (Switzerland) and expanded under standard cell culture conditions (37°C and 5% CO2). The medium is completely replaced every two to three days. All cells are passaged at 90% confluence and used for bioprinting at a maximum passage number of 4. hASCs and HllVECs are thawed and expanded in Endothelial Cell Growth Medium 2 (ECGM 2 - PromoCell, Germany) supplemented with 100 μl / mL penicillin and 100 μg / mL streptomycin (Gibco, USA). HDFs are thawed and expanded in Dulbecco's Modified Eagle's Medium with 4.5 g / mL glucose (DMEM high glucose - Corning, USA) supplemented with 10% v / v fetal calf serum (Corning, USA) as well as 100 lU / mL penicillin and 100 pg / mL streptomycin (Gibco, USA).

[0095] Preparation of the photopolymerizable or photocrosslinkable liquids with the prepared cell cultures: A total of two different photopolymerizable or photocrosslinkable liquids are prepared, each mixed with the cell cultures cultivated in the previous step. The first liquid should contain the hASCs and the hDFs, and the second liquid should contain the hllVECs. These liquids are prepared by dissolving and subsequently diluting the respective lyophilized material in PBS. The cells are then mixed directly into the photopolymerizable or photocrosslinkable liquids shortly before printing. The compositions of the resulting liquids are listed in Table 1.

[0096] Table 1: Composition of the photopolymerizable or photocrosslinkable fluids. For the various fluids used to manufacture each compartment of the breast implant, the components and concentrations are given in wt.%. Cell types and concentrations for cell-loaded photopolymerizable or photocrosslinkable fluids are given.

[0097] LAP: lithium phenyl 2,4,6-trimethylbenzoylphosphinate

[0098] 3D bioprinting of the 3D adipose tissue scaffold as a breast implant

[0099] Three-dimensional 3D adipose tissue scaffolds are designed using Rhinoceros 6 (Robert McNeel & Associates, Seattle, USA). The STL file is exported, and the projection masks are created by the bioprinter's software. Bioprinting is performed using a bioprinting platform from Cellbricks GmbH, as described in EP 3 018 531 A1. The printed 3D adipose tissue scaffolds are embedded in a soft hydrogel by overlaying them with 400 pL of the encapsulating gel in 24-well plates and post-curing for 10 minutes with a 385 nm light source (2.5 mW / cm2). 500 pL of ECGM2 with 75 U / mL hyaluronidase (STEMCELL Technologies, Canada) are added to the wells of the plate and incubated overnight under cell culture conditions to allow enzymatic digestion of the interior of the prepared channel structures, leading to the release of the embedded HLIVECs and their subsequent adhesion to the channel walls.

[0100] In situ differentiation of the 3D adipose tissue scaffold

[0101] After printing, the fabricated 3D adipose tissue scaffolds are cultured in specialized adipogenic culture media to form adipose tissue in situ. They are first acclimated in ECGM2 for 3 days. The prints are then cultured in adipogenic induction medium based on ECGM2 supplemented with 0.25 mM isobutyl-1-methylxanthine (IBMX), 0.1 pM insulin, 1 pM rosiglitazone, 0.2 nM triiodothyronine, and 1 pM dexamethasone. Following this, the medium is completely changed to adipogenic differentiation medium. This medium has the same composition as the induction medium but does not contain IBMX. The printed 3D adipose tissue scaffolds are cultured in adipogenic differentiation medium for the entire remaining culture period (up to 4 weeks after printing), with complete daily medium changes.

[0102] Bioanalytical methods and results: a) The 3D bioprinted breast implant model is vital throughout the entire culture period of 4 weeks and shows high cellular dynamics:

[0103] Live-to-dead staining (calcein-AM, Hoechst 33342, and propidium iodide) of the bioprinted adipose tissue models and fluorescence microscopy show a high proportion of live cells and a low content of dead cells, respectively, 3, 13, and 27 days after printing. In addition to qualitative detection, live and dead cell populations are quantified, yielding >90% live cell content in the bioprinted adipose tissue models at all analysis time points. Vitality can be indirectly demonstrated by high cellular activity in the printed constructs. Non-invasive automated microscopy reveals dynamic cell behavior with migrating and interacting cell clusters and the formation of multicellular tissue-like structures. In particular, cell outgrowth from the bioprinted constructs into the surrounding cell-free, embedding hydrogel is observed.Embedding the bioprinted adipose tissue models in a hydrogel that simulates the ECM (collagenoids and hyaluronic acid as material basis) and biomechanics (similar substrate stiffness) of adipose tissue offers the opportunity to utilize an in vitro setup, which is relevant for later implantation into native adipose tissue.

[0104] Experimental Procedure: Differentiation medium is used as the base for staining. Calcein-AM and Hoechst 33342 are diluted 1:5000 (0.1 μl per 500 μl), while PI is used at a dilution of 1:50 (10 μl per 500 μl). Staining is performed in a 24-well plate. 500 μl of this staining solution is applied to a bioprinted adipose tissue model. Incubation takes place at room temperature with agitation and lasts three hours, with the staining solution being resuspended hourly. The live-dead staining is photographed using an automated fluorescence microscope (NYONE®, SynenTec) and objectified for staining intensity using YT-Software®. This is used for quantification of live and dead cell populations. b) The 3D bioprinted adipose tissue contains adipocytes after sufficient maturation and is vascularized:

[0105] Adipose tissue precursors created by 3D bioprinting can be successfully matured into mature adipose tissue using a customized differentiation protocol, which is characterized by the presence of adipocytes. Printed adipose stem cells (hASCs) are differentiated into adipocytes by culture in an adipogenic medium, detectable by the appearance of cellular adipose vesicles, the number of which increases with progressing culture. This process can be demonstrated by light microscopy and fat-specific Nile Red staining in control cultures in Petri dishes as well as in the bioprinted adipose tissue. After several weeks of culture, the printed endothelial cells (HUVECs) can be detected not only in the predefined, 3D-printed channel system, but also develop capillary-like networks that permeate other parts of the bioprinted adipose tissue.This is demonstrated by live cell staining and specifically by anti-CD31 staining in the bioprinted adipose tissue, even after several weeks of culture. Adipose and vascular tissue structures can also be demonstrated by scanning electron microscopy and histologically. Specifically, stained thin sections of the mature bioprinted adipose tissue present a loose, fat vacuole-rich tissue structure not only within the constructs but also surrounding the embedding hydrogel. Immunohistochemical detection of mature adipocytes (anti-S100 staining), endothelial cells (anti-CD31 staining), and the formation of new cell-derived extracellular matrix (Masson-Goldner trichrome staining) confirms the maturation process of the bioprinted constructs into vascularized adipose tissue.

[0106] Experimental implementation:

[0107] Nile Red staining: Nile Red staining is used to objectively determine the number of adipose vesicles and to visualize them. Nile Red is a fluorescent phenoxazine dye (excitation: 515-560 nm; emission: >590 nm) that, due to its lipophilicity, can stain adipose vesicles. An adipose tissue print is transferred to a 24-well plate and initially incubated for 30 minutes at room temperature in 10% formalin solution for fixation. The print is then washed with 500 μl of PBS for 10 minutes, after which it is permeabilized for another 10 minutes in 500 μl of 0.1% Triton X-100 solution. After another PBS wash, the print is incubated for 30 minutes at room temperature in the Nile Red staining solution (0.25 mg / ml in pure ethanol). Following a final wash step with PBS, a 10-minute incubation at room temperature with DAPI (4',6-diamidine-2-phenylindole; 10 pg / ml in PBS) is carried out to counterstain the cell nuclei.

[0108] The Nile Red fat stain is also photographed using the automated fluorescence microscope NYONE® (SynenTec, excitation: 562 nm (lime), emission: 628 nm (red)) and objectified by the YT-Software® with regard to staining intensity and extent.

[0109] Sample preparation for (immuno)histochemical staining: A fat tissue print is transferred to a 24-well plate and initially incubated for 30 minutes at room temperature in 10% formalin solution for fixation. The print is then washed with 500 μl of PBS for 10 minutes before being transferred to a sample cassette for dehydration through an ascending alcohol series (50% ethanol, 70% ethanol, 96% ethanol, xylene, 5 minutes each). The dehydrated print is embedded in paraffin, and the resulting block is sectioned using a microtome. The resulting thin sections are mounted on glass slides, air-dried, and stored in a cool, dark place until staining.

[0110] Immunohistochemical staining: The dehydrated thin sections are rehydrated using a descending alcoholic series, a subsequent wash step with ultrapure water, and a final incubation in PBS. Subsequently, they are permeabilized with a PBS + 0.25% Triton X-100 wash step (10 min), and the samples are overlaid with 10% goat serum in PBS (30 min at room temperature) to block nonspecific antibody binding. Staining solutions containing one of the two primary antibodies (mouse) against human CD31 or S100 are prepared in PBS + 10% goat serum and applied to the thin sections overnight at 4°C. This is followed by a complete exchange with the secondary antibody solution (goat anti-mouse, conjugated with horseradish peroxidase), followed by a further incubation step at room temperature for 1 h.Staining is completed by incubating with a chromogenic substrate solution (3,3'-diaminobenzidine) at room temperature for 10 minutes, followed by rinsing with tap water for 5 minutes. Immunohistochemical staining is complemented with a hematoxylin counterstain. For this purpose, the thin sections are incubated with a Mayer's hematoxylin solution for 8 minutes at room temperature, then rinsed with tap water for 10 minutes and briefly again with ultrapure water. Finally, the stained thin sections are dehydrated using an ascending alcoholic series and mounted with a non-aqueous medium.

[0111] Masson-Goldner trichrome staining: The dehydrated thin sections are rehydrated using a descending alcoholic series followed by a wash step with ultrapure water. They are then incubated with Weigert's hematoxylin solution for 15 minutes at room temperature, then rinsed with tap water for 8 minutes and briefly rinsed again with ultrapure water. The thin sections are then stained with an acid fuchsin-Ponceau 2R-azophloxine solution for 4 minutes before being immersed in 1% acetic acid for a brief color differentiation step. This is followed by a 30-minute staining with phosphomolybdic acid-orange G solution, followed by a brief differentiation step in 1% acetic acid. The final staining step involves incubation with light green solution followed by differentiation in 1% acetic acid. The stained thin sections are washed with running tap water for 1 min and finally dehydrated with an ascending alcoholic series.Finally, the specimen is covered with a non-aqueous mounting medium.

[0112] Figures 1a to 1c show the differentiation of hASCs into adipocytes (control group in 2D culture). Successful generation of adipocytes is evident by strong cell morphological changes, particularly the formation of intracellular fat vesicles, which microscopically manifest as oil vesicles. With increasing culture time under adipogenic conditions, there is increased fat vesicle formation and the development of larger vesicles, which is a characteristic of mature adipocytes. Images from different time points in culture: 3 days (left), 6 days (middle), and 10 days (right) after the start of differentiation. Scale bar: 100 pm.

[0113] Fig. 2a and 2b show histological and electron microscopic evidence of formed adipose tissue. Contrast staining with hematoxylin and eosin (left) of bioprinted adipose tissue after 4 weeks in culture shows a loose, fat vacuole-rich tissue with interspersed connective tissue. Scale bar: 100 pm. Scanning electron microscopy examination of the ultrastructure of the bioprinted adipose tissue (right) reveals cellular components and, in particular, prominent fat vesicles (light gray, arrows) embedded in the printed matrix (hydrogel, dark gray). Scale bar: 10 pm.

[0114] Fig. 3a to 3c show the vascularization of the bioprinted adipose tissue. Endothelial cells used in bioprinting line the printed channels, thus forming larger vessel-like structures, but also arrange themselves into microcapillary structures over the culture period. Immunohistological detection of structures formed by endothelial cells using anti-CD31 staining in the printed channel (left, cross-section, cell-lined channel wall marked with arrows), as well as in formed microcapillary cell clusters (center) in the bioprinted adipose tissue after 4 weeks of culture. Scale bar: 200 pm. Scanning electron micrograph of a transverse section of the bioprinted adipose tissue model (right) reveals cross-sections of printed channels (marked with white arrows). Scale bar: 500 pm.

Claims

A 3D adipose tissue scaffold for use as a breast implant for the surgical, therapeutic, prophylactic, or aesthetic reconstruction, reconstruction, or replacement of human breast tissue, wherein the 3D adipose tissue scaffold comprises a matrix made of a biocompatible polymer populated with a plurality of biological cell types, wherein the population with the plurality of cell types takes place during the construction of the matrix made of biocompatible polymer with the formation of a blood vessel structure network within the 3D adipose tissue scaffold, characterized in that light-based, structuring curing of one or more photopolymerizable or photocrosslinkable liquids is used for the construction of the matrix made of biocompatible polymer. The 3D adipose tissue scaffold according to claim 1, wherein the human breast tissue has been removed for the prevention of, or as a result of, a disease.The 3D adipose tissue scaffold of claim 1 or 2, wherein the disease is breast cancer. The 3D adipose tissue scaffold of any one of claims 1 to 3, wherein the cells of the plurality of cell types are autologous cells. The 3D adipose tissue scaffold of any one of claims 1 to 4, wherein the cells of the plurality of cell types are selected from the group consisting of adipocytes, adipocyte stem / progenitor cells, fibroblasts, endothelial cells, and a combination thereof. The 3D adipose tissue scaffold of claim 5, wherein the cells of the plurality of cell types are adipocytes and / or adipocyte stem / progenitor cells, and fibroblasts and / or endothelial cells. The 3D adipose tissue scaffold of any one of claims 1 to 6, wherein the light-based patterning curing is a stereolithographic 3D printing process. The 3D adipose tissue scaffold according to claim 7, wherein the stereolithographic 3D printing process is a projection-based exposure process. The 3D adipose tissue scaffold according to any one of claims 1 to 8, wherein a first photopolymerizable or photocrosslinkable liquid containing adipocyte stem cells and a second photopolymerizable or photocrosslinkable liquid containing endothelial cells or fibroblasts are used for the light-based, structuring curing. The 3D adipose tissue scaffold according to claim 9, wherein the second photopolymerizable or photocrosslinkable liquid contains endothelial cells. The 3D adipose tissue scaffold according to claim 10, wherein a third photopolymerizable or photocrosslinkable liquid containing fibroblasts is additionally used for the light-based, structuring curing. The 3D adipose tissue scaffold according to claim 10, wherein the first photopolymerizable or photocrosslinkable liquid additionally contains fibroblasts.3D adipose tissue scaffold according to one of claims 9 to 12, wherein one or more messenger substances are further used to differentiate the adipocyte stem cells into adipocyte progenitor cells and adipocytes. 3D adipose tissue scaffold according to one of claims 1 to 13, wherein a photoblocker is used in one or more of the one or more photopolymerizable or photocrosslinkable liquids. Use of a 3D adipose tissue scaffold as a breast implant in humans, wherein the 3D adipose tissue scaffold has a matrix made of a biocompatible polymer that is populated with a plurality of cell types, wherein the colonization with the plurality of cell types takes place during the construction of the matrix made of biocompatible polymer with the formation of a blood vessel structure network within the 3D adipose tissue scaffold, characterized in that light-based,. structuring curing of a photopolymerizable or photocrosslinkable liquid is used.

Images