Mould and method for tissue graft manufacturing

Abstract

The present invention relates to a mould (1), the mould comprising a receptacle (10) and a lid (20) for forming a tissue graft, such as an autologous tissue graft for transplant and suturing. The mould is adapted to contain layers of biological material, providing pressure to the biological material and drain out excess fluid, thus providing a fast and effective means for building a tissue graft. The mould may comprise one or more resilient members, and at least one or more of the resilient members (RM) may be suitable to provide a specified force to apply the desired pressure to the tissue graft. The invention further relates to a method of using the mould.

Description

[0001] 85186PC01

[0002] 1

[0003] MOULD AND METHOD FOR TISSUE GRAFT MANUFACTURING

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to forming tissue grafts. In particular the present invention relates to a mould for forming tissue grafts, a method of providing a tissue graft using a mould, and a kit of parts for forming tissue grafts.

[0006] BACKGROUND OF THE INVENTION

[0007] Tissue engineering has emerged as a promising field aimed at developing biological substitutes to restore, maintain, or improve tissue function. One of the critical components in tissue engineering is the creation of tissue grafts, which are used to replace or repair damaged tissues. Tissue grafts can be derived from various sources, including autologous tissue (tissue from the patient's own body), allogeneic tissue (tissue from a human donor), xenogeneic tissue (donor tissue from another species), and synthetic materials. These grafts are essential in various medical applications, such as reconstructive surgery, wound healing, and organ transplantation.

[0008] The process of manufacturing tissue grafts involves several steps, including the selection of appropriate materials, the creation of a scaffold to support cell growth, and the integration of cells into the scaffold. The scaffold must be biocompatible, provide mechanical support, and promote cell adhesion and proliferation.

[0009] One of the primary challenges in the preparation of autologous tissue grafts is the expansion of cells. This primarily takes place in a laboratory setting, removed from the patient, and in highly regulated, time consuming and thus expensive procedures. Current methods of layering tissue grafts are time consuming and do not guarantee a consistent result.

[0010] Hence, an efficient device and method of preparing a tissue graft, in a timely manner, beside the patient in an operating theatre, would be advantageous. 85186PC01

[0011] 2

[0012] OBJECT OF THE INVENTION

[0013] The present invention addresses these challenges by providing an improved method and device for manufacturing tissue grafts, in particular autologous tissue grafts. The invention includes a mould designed to shape biological material into a tissue graft, ensuring that the grafts have the desired mechanical properties, shape, thickness, and biocompatibility, by providing a consistent and finely tuned pressure, while being effective to work with.

[0014] It is a further objective of the present invention to provide an alternative to the prior art.

[0015] SUMMARY OF THE INVENTION

[0016] Thus, the above-described object and several other objects are intended to be obtained in a first aspect of the invention by providing a mould adapted to shape a biological material into a tissue graft, the mould comprising:

[0017] -a receptacle comprising one or more peripheral sidewall(s) having an initial height hl and a bottom surface having an area al, said sidewall(s) and bottom surface defining an inner compartment for containing the biological material during use of the mould, the one or more peripheral sidewall(s) being adapted to fold or collapse to a final height h2 matching a thickness of the tissue graft, during use of the mould, wherein h2 > 0.5 mm and h2 < hl, and

[0018] -a lid adapted to fit onto the receptacle to close the inner compartment, wherein the lid and the receptacle are manufactured from a biocompatible material; such as a polymer material, a metal material, a cellulose-based material, or a combination thereof, wherein the one or more peripheral sidewall(s) is / are adapted to fold or collapse when a force F is applied to the mould during use, the force F moving the lid and the bottom surface towards each other to create a pressure P, defined by F / al, 85186PC01

[0019] 3 which pressure is thereby applied to the biological material during shaping of the tissue graft, and wherein the one or more peripheral sidewall(s) are liquid-tight, and the lid and / or any air gap arising between the sidewall(s) and the lid, after fitting the lid onto the receptacle during use, are configured so that fluid can be drained from the inner compartment only therethrough.

[0020] In the above description of the mould, it should be understood that the material should be biocompatible. The materials listed are all presented as examples of possible materials.

[0021] A metal material may e.g. be stainless steel, aluminium, or titanium. A non- exhaustive list of possible metal materials include Steel 316L, Steel 304, Titanium alloys such as Ti-6AI-4V, Nitinol (NiTi), and Tantalum.

[0022] A cellulose-based material may e.g. be cardboard. In such embodiments, the cellulose-based material may be provided with a coating to ensure that the mould is liquid-tight and does not absorb material being arranged in the mould during use.

[0023] The feature that "the force F is moving the lid and the bottom surface towards each other" may mean that only one of the lid or the bottom surface of the receptacle is moving whereas the other is stationary, such as resting on a table. As will be described in relation to the figures, this is typically the case. Alternatively, it may mean that both the lid and the bottom surface are moving.

[0024] Since the pressure is applied to the biological material, which is contained in the inner compartment, it may also be referred to as "internal pressure". The intended meaning of this term is that it should not be read as a hydrostatic pressure inside a fluid-filled enclosure even though a similar term is often used for that.

[0025] In some embodiments of the invention, the mould is adapted to sustain a pressure P of at least between 325 Pa and 660 Pa during use. By "at least" is 85186PC01

[0026] 4 preferably meant that the sidewall(s) should be configured to withstand any pressure within this range, but it does not exclude that the sidewall(s) may also be able to withstand pressures outside this range. During the development of the present invention, this pressure range is found to be particularly advantageous for ensuring that the correct pressure is applied to the tissue graft in order to obtain the desired properties thereof. These tests were performed by arranging different weight members of known mass on top of the mould to apply a thereby known force F. Different weight members were tested and the resulting tissue crafts were analysed to determine satisfactory ranges. It is to be understood, that applying too high pressure will force fluid from the tissue graft at a pace potentially destroying the graft, and too low pressure does not ensure proper drainage of excess fluid from the graft. Therefore a too high or too low pressure may cause the surfaces of the graft to be uneven, not suitable for the following transplantation.

[0027] In a more preferred embodiment, the pressure is between 400 and 600 Pa.

[0028] By stating that the mould is adapted to sustain a certain pressure, it is preferably meant that the mould should not break, such as crack, during use of the mould. It is acceptable that the sidewall(s) bulge out sideways as long as it does not negatively impact the function of the mould.

[0029] The force may be applied in any suitable manner of which different examples will be described below and illustrated in the figures. These examples are non-limiting to the broadest scope of protection. The force could e.g. be applied by arranging an object of specified mass on top of the closed mould, or the force could be applied by another external device, such as automated equipment.

[0030] The invention is particularly, but not exclusively, advantageous for providing a device adapted for fast and effective shaping / building of a tissue graft for a patient. In particular, the invention is advantageous when preparing an autologous tissue graft, as biologic material from the patient is to be obtained to prepare the tissue graft. 85186PC01

[0031] 5

[0032] In the context of the present invention, it is to be understood, that the height of the sidewalls is adapted to reduce, when force is applied to the mould, and the reduced height ensures that pressure is applied to the tissue graft during preparation. The pressure ensures that excess fluid is drained from the tissue graft, thus providing a tissue graft which can be handled during graft transplantation.

[0033] It is further to be understood, that the lid and the bottom surface are adapted so as to be prevented from abutting, which would spoil the tissue graft, i.e. squeeze the graft too much. In other words, the mould is adapted as to provide a gap between the bottom surface of the receptacle and the lid when pressure is applied and the tissue graft is drained of excess fluid. This gap preferably corresponds to the height h2. The mould is adapted to provide a gap between 0.5 mm and 5 mm, preferably between 1 mm and 4 mm, even more preferably between 1 mm and 3 mm.

[0034] In the context of the present invention, it is to be understood, that the mould should in some embodiments sustain at least between 325 and 660 Pa without the lid or the bottom surface buckling, bulging or coming apart during pressure application.

[0035] It is further to be understood, that the sidewalls of the receptacle are advantageous for retaining the fluid elements of the tissue graft, prior to a heating process which is typically used for curing the layers of the graft, prior to applying pressure to said graft.

[0036] Even further, it is to be understood, that when an autologous tissue graft is prepared, a heating process is typically applied, and thus the mould is adapted to be stable during said heating process. In presently preferred embodiments, the mould must be stable at least between 20 and 41 degrees Celsius.

[0037] It is further to be understood, that the bottom surface may be made in any shape, such as round, and wherein the receptacle thus only has a single, peripheral sidewall. It is further to be understood, that the lid is to be made in a shape corresponding to the shape of the bottom surface. 85186PC01

[0038] 6

[0039] The tissue graft could also be used for in vitro tissue culturing aiming at research studies and pharmaceutical testing in a 3D culture system.

[0040] In other embodiments, the bottom surface of the receptacle may be shaped as a polygon with three or more sides, such as a triangle, a rectangle, a square, a pentagon, a hexagon, a heptagon, an octagon, a nonagon or a decagon. It is to be construed such that any shape fitting for the tissue graft position, on or in a patient, may be provided by the mould.

[0041] Additionally, it is advantageous that the mould is made from a biocompatible material, such as a polymer, ensuring that the tissue graft remains uncontaminated and safe for implantation.

[0042] In some embodiments, the mould is manufactured from a material which is suitable for sterilization, such as using alcohol or autoclaving.

[0043] In a preferred embodiment, the lid and the receptacle are attached to each other by a hinge, the receptacle and lid adapted to fold together thereby defining the closed inner compartment. This embodiment provides for an easy and convenient method of closing the lid onto the receptacle, ensuring that any fluids are not spilled from the receptacle, prior to heating. Further, this embodiment is advantageous for ensuring that the lid is not misplaced during preparation of the tissue graft.

[0044] In another preferred embodiment, the lid and receptacle may be made in one piece, such as moulded in one singular piece. This embodiment is particularly advantageous for providing a fast and cost-effective device for the production of moulds. Even further, this embodiment is particularly advantageous for ensuring easy cleaning of the mould, after use, as no crevasses or other voids are present in the mould.

[0045] In an embodiment, wherein the mould is moulded in one piece, the mould may be manufactured by being subjected to an initial moulding process, after which, the 85186PC01

[0046] 7 mould would be postprocessed by thermoforming to create rigid and resilient portions of the mould.

[0047] In some embodiments, the one or more peripheral sidewalls of the mould may be pleated. This embodiment is particularly advantageous for providing a predetermined compression / collapsibility of said one or more sidewalls.

[0048] In yet another preferred embodiment comprising a hinge between the lid and the receptacle, the mould further comprises a necking section extending between the lid and the receptacle, the receptacle and lid adapted to fold together. This embodiment is particularly advantageous for ensuring that the hinge folds in a predetermined manner thereby facilitating the closing of the mould.

[0049] In an advantageous embodiment, the mould further comprises a locking mechanism adapted to secure the lid to the receptacle. This embodiment is advantageous for ensuring that the lid stays in a predetermined position during the application of pressure.

[0050] In another preferred embodiment, the locking mechanism comprises a snap lock. This embodiment ensures easy handling of the mould.

[0051] In yet another preferred embodiment, the locking mechanism comprises first and second locking members, the locking members protruding from the receptacle and being adapted for receiving the lid from a side of said receptacle. This embodiment is particularly advantageous for ensuring that the lid is positioned correctly on the receptacle prior to application of pressure.

[0052] In a preferred embodiment, the mould further comprises a resilient member adapted to provide the force F between the lid and the bottom surface of the receptacle during use of the mould.

[0053] It is to be understood, that in order to ensure a desired pressure being applied to the biological material, and since F = P x al, a mould having a larger area al requires more force from the resilient member. 85186PC01

[0054] 8

[0055] In some embodiments, the resilient member may be a spring or elastic element, adapted to sustain an even pressure to the graft. Different examples of resilient members will be shown in the figures.

[0056] In another preferred embodiment, the resilient member is comprised in the locking mechanism. This embodiment is particularly advantageous for providing a mould which is easier to use and provides a method of preparing a tissue graft with a low risk of failure.

[0057] In yet another preferred embodiment, at least a portion of the one or more peripheral sidewall(s) of the receptacle is resilient. This embodiment may be used to provide a predetermined collapse of the sidewalls during the application of pressure.

[0058] In other preferred embodiments, the sidewalls are corrugated to facilitate the reduction from hl to h2 during use of the mould.

[0059] In an advantageous embodiment, the lid further comprises at least one through hole, such as a plurality of through holes. Such through holes are used for draining fluid from the biological material thereby obtaining a tissue graft with the required properties. A plurality of through holes are preferably spaced apart in a manner that ensures a uniform compression of the biological material and thereby also a uniform drainage of excess fluid during use of the mould. Furthermore, such holes will provide a pressure equalization and thereby prevent underpressure arising in the inner compartment when the mould is opened again to remove the tissue graft. Thereby the risk of damaging the tissue graft during the opening of the mould is minimized.

[0060] The above embodiments are particularly advantageous for ensuring correct draining of excess fluids during the application of pressure.

[0061] In some embodiments of the invention, the bottom surface is provided with markings, such as grid-shaped markings, configured to guide the manual placement of biological material and thereby ensure a predetermined arrangement of the biological material during use of the mould. As will be described in further 85186PC01

[0062] 9 details below, the mould will typically be used with minced biological material. Therefore, a small piece of biological material can be placed e.g. in each space of the grid. The grid may be visual markings or it may be in the form of slightly dented lines on the bottom surface. By ensuring a uniform arrangement of the biological material, a more efficient growth process and subsequent healing can be obtained.

[0063] In some embodiments of the invention, the mould is provided with one or more protrusions dimensioned and arranged to ensure that the lid and the bottom surface will not be moved closer to each other than to a distance matching the final height h2 of the one or more sidewall(s).

[0064] During use of the mould, the amount of biological material is chosen to ensure the desired thickness and density of the final tissue graft when taking into account the dimensions of the inner compartment. The use of a mould with one or more protrusions makes it easier to ensure that the desired thickness of the tissue graft is obtained.

[0065] In a preferred embodiment, the mould further comprises an intermediary sheet adapted for insertion between the bottom surface of the receptacle and the lid.

[0066] In another preferred embodiment, the intermediary sheet has an area a2, wherein a2 < al.

[0067] In yet another preferred embodiment, the intermediary sheet is a biocompatible mesh. It may e.g. be polyester-based as is typically used for absorbable sutures and surgical meshes. Such a kind of mesh will be known on its own by a person working within this field.

[0068] The intermediate sheet may further be selected from acellular auto- / allo- / or xenogenic biological matrices, such as a cellular small intestinal submucosa, acellular dermis or acellular bladder matrix.

[0069] In yet another preferred embodiment, the intermediary sheet has a thickness of between 0.01 and 3 mm. 85186PC01

[0070] 10

[0071] It is to be understood, that the intermediary sheet is provided as a structural layer, such as a scaffold, and wherein biological material and ECM is absorbed, fused, or intertwines with the intermediary sheet, thus providing for a structurally stable tissue graft, suitable for transplantation and suturing, i.e., typical handling during surgery. The intermediary sheet may further be used to separate different types of cells thereby facilitating the growth of a tissue graft with layers of different types of cells, such as skin cells and muscle cells.

[0072] In yet other embodiments, there may be two or more intermediary sheets. It is to be construed, that the two or more intermediary sheets may be from different materials.

[0073] In an advantageous embodiment, portions of the one or more peripheral sidewall(s) of the receptacle comprise recesses, the recesses extending radially inwards, towards the bottom surface. This embodiment is particularly advantageous for guiding the lid onto the receptacle, ensuring the correct position of the lid, and furthermore serves to prevent sideways movement of the lid, relative to the receptacle, during the application of pressure.

[0074] In other advantageous embodiments, portions of the lid extend radially inwards, providing the same advantages as stated above. Further, in some embodiments, both the lid and the sidewalls of the receptacle are adapted with corresponding recesses to improve the guidance of the lid into the correct position and to maintain said position during the application of pressure.

[0075] In another advantageous embodiment, a top surface of the lid extends radially outwards, defining a lip. This embodiment may be particularly advantageous for ensuring a predetermined drainage of excess fluid from the mould. Furthermore, the lip may ensure that the lid does not extend completely into the receptacle, eliminating the gap which is needed to prevent spoiling of the tissue graft during the application of pressure. Alternatively or in combination therewith, the lid and / or the receptacle may be provided with one or more stops, such as protrusions or other mechanical engagement members, configured to ensure that 85186PC01

[0076] 11 the final distance between the lid and the bottom surface is as required. This final distance corresponds to the final height, h2, of the one or more sidewall(s).

[0077] In yet another advantageous embodiment, at least one of the receptacles or the lid is made from a biocompatible material selected from one or more of: silicone, thermoplastic polyurethane TPU, polyethylene PE or polypropylene PP.

[0078] Other materials of the receptacle and the lid may be selected from one or more of:

[0079] • Polylactic Acid (PLA)

[0080] • Polycaprolactone (PCL)

[0081] • Polyethylene Glycol (PEG)

[0082] • Silicone

[0083] • Polyvinyl Alcohol (PVA)

[0084] • Polyhydroxyalkanoates (PHA)

[0085] • Polyglycolic Acid (PGA)

[0086] • Poly(L-lactide-co-glycolide) (PLGA)

[0087] • Poly(ethylene oxide) (PEO)

[0088] • Poly(ethylene terephthalate) (PET)

[0089] • Poly(ether ether ketone) (PEEK)

[0090] • Poly(2-hydroxyethyl methacrylate) (PHEMA)

[0091] • Poly (£-ca prolactone) (PCL)

[0092] • Poly(ethylene-co-vinyl acetate) (PEVA)

[0093] • Poly(ethylene glycol) diacrylate (PEGDA)

[0094] • Poly(ethylene glycol) dimethacrylate (PEGDMA)

[0095] An alternative list of possible polymer materials, partly overlapping with the previous list, is: Medical-grade polycarbonate (PC), Polyethylene (HDPE, UHMWPE), Polypropylene (PP), Polyethylene terephthalate (PET, PETG), Polystyrene (PS), Acrylics (PMMA), Polyetheretherketone (PEEK), Polyurethane (PU), Silicone rubber (LSR, HTV), Nylon (PA 6, PA 12), ABS (with medical grades), and PVC (medical-grade DEHP-free).

[0096] As described above, the receptacle and the lid may also be made from a cellulose- based material, such as cardboard. 85186PC01

[0097] 12

[0098] In a preferred embodiment, hl is between 5 and 30 mm, preferably between 5 and 20 mm. This embodiment is advantageous for reducing waste, as the mould is dimensioned for the tissue graft and further may provide an indication to a user, that if the materials provided to the mould does not fill the receptacle, or the receptacle overfills, something has not been performed correct. Further, the receptacle may be provided with lines adapted to display correct fill levels for different layers of the tissue graft. In some embodiments, more extracellular matrix (ECM) or biological material may be required, and wherein hl may be between 5 and 35 mm, such as between 10 and 30 mm or such as between 15 and 25 mm. More details about the ECM will be given below.

[0099] In another preferred embodiment, the lid comprises one or more supports, the supports extending perpendicular or substantially perpendicular to a main plane of the lid. It is to be understood, that the one or more supports extends in a direction opposite to the receptacle, when the lid is positioned on top of the receptacle. This embodiment is particularly advantageous for ensuring that excess fluids are enabled to drain from the receptacle, when the receptacle is turned upside down, such as after heating the mould, as the distance provides a gap between the lid and a surface, such as when positioned on a table.

[0100] In yet another preferred embodiment, one or more inner surfaces of the mould is / are coated with a biocompatible or bioactive material.

[0101] The biocompatible coating may be selected from one or more of the following :

[0102] • Polycaprolactone (PCL)

[0103] • Polylactic-co-glycolic acid (PLGA)

[0104] • Polyethylene glycol (PEG)

[0105] • Hydroxyapatite

[0106] • Chitosan

[0107] • Collagen

[0108] • Gelatin

[0109] • Hyaluronic acid

[0110] • Poly(D,L-lactic acid) (PDLLA)

[0111] • Polyvinyl alcohol (PVA) 85186PC01

[0112] 13

[0113] The bioactive coating may be selected from one or more of the following:

[0114] • Hyaluronic acid

[0115] • Chitosan

[0116] • Collagen

[0117] • Gelatin

[0118] • Hydroxyapatite

[0119] • Bioactive glass

[0120] • Calcium phosphate

[0121] • Alginate

[0122] • Fibrin

[0123] • Silk fibroin

[0124] It is further to be understood that one or more of the biocompatible and bioactive coatings may be combined.

[0125] This embodiment is particularly advantageous for providing an optimal environment to mould the tissue graft within. In particular, autologous tissue grafts require optimal conditions to ensure proper graft acceptance or graft integration. Such a coating may e.g. be used to ensure that the material of which the mould is made does not negatively impact the tissue graft. It may also be used to improve the heating properties of the mould, and / or it may facilitate the removal of the tissue graft from the mould at the end of the process.

[0126] In some embodiments of the invention, the mould is used together with a tray in which the mould is to be arranged during the application of the force. Such a tray can then collect the fluid being drained from the biological material, so that the fluid can easily be poured into a drain or a separate container for later disposal.

[0127] In a second aspect, the invention relates to method of preparing a tissue graft from a biological material, the method comprising the following steps:

[0128] -providing the mould according to the first aspect of the invention, -layering the biological material by the following steps:

[0129] -step 1 : providing biological material to the receptacle and / or the intermediary sheet, when present, 85186PC01

[0130] 14

[0131] -step 2: providing an extracellular matrix material to the receptacle and / or the intermediary sheet, when present,

[0132] -step 3: when using an intermediate sheet, inserting the intermediary sheet within the receptacle; wherein at least one of step 1 and step 2 may be performed in any order and / or repeated prior to or after step 3; and, after layering the biological material, performing the following further steps:

[0133] -step 4: supplying energy to the mould containing the biological material in order to provide a curing of the biological material, -step 5: positioning the lid on the receptacle, and

[0134] -step 6: applying a force F to the mould thereby applying pressure P, such as pressure being between 325 Pa and 660 Pa, to the layered biological material to shape it into a tissue graft.

[0135] The extracellular material, ECM, is liquid when being added to the biological material arranged in the mould, and it typically fully covers the biological material.

[0136] In some embodiments of the invention, step 4 is performed by providing heat to the receptacle for at least 30 seconds. In alternative embodiments, step 4 is performed by a UV radiation step or an ultrasound step. Such an embodiment may be particularly advantageous to ensure, that health professionals have the equipment needed to prepare the tissue graft, i.e., if heat is not available, one or more of the ECM or buffers may be replaced with an ingredient suitable for curing by either ultraviolet radiation (UV), light curing, chemical curing, enzymatical treatment or ultrasound.

[0137] The method is particularly advantageous for preparing a biological graft, and the method steps ensure a uniform, consistent result, which is easy to replicate, thus ensuring consistent high quality, correct structural integrity of the tissue graft, low spillage of ECM, and reduced waste as the moulds can be reused post sterilization or recycled.

[0138] In the context of the present invention, biological material may be cells or a tissue sample from a patient in need of a tissue graft. In some embodiments of the invention, the biological material has been prepared by mechanical dissection and 85186PC01

[0139] 15 may then be referred to as "minced". When a tissue graft is prepared and transplanted, the cells grow from outer surfaces of the biological material, and therefore an advantage of using minced material is that the surface area from which the cells can grow is significantly larger for a given amount of material than if one coherent piece of material is used. The ECM may function as a scaffold for cellular migration and / or as a growth medium for the biological material and may contain growth factors or other added factors to promote healing. In embodiments of the invention, wherein minced biological material is used, the ECM also assists in binding the small pieces of biological material together.

[0140] The use of minced biological material for the tissue graft compared to using a suspension of cells, as in some alternative methods, is that a mechanical handling of the biological material is sufficient and a more efficient process is obtained, whereby the duration of time in which the material is removed from the body of the patient is lowered and the use of foreign enzymes and chemicals is reduced. This limits the time needed, lowers the risk of damage to the material to be transplanted, and lowers the costs of creating the graft.

[0141] In a preferred embodiment, the method further comprises the following step, after step 5: -turning the mould upside down, the lid facing downwards, to drain any fluid from within the receptacle. This embodiment is particularly advantageous, as this speeds up the preparation time of the biological graft, while providing the correct pressure within the receptacle.

[0142] In another preferred embodiment, step 5 may be performed prior to step 4.

[0143] In yet another preferred embodiment, the pressure is provided for between 1 minute and 20 minutes, preferably for between 2 and 15 minutes, even more preferably for approximately 2 to 5 minutes. Current studies have demonstrated that approximately 5 minutes of pressure of approximately 450 Pa provides for a structural integrity of the tissue graft, while providing smooth surfaces of the graft which are easy to work with during transplantation and suturing, i.e. surgical handling. 85186PC01

[0144] 16

[0145] In an advantageous embodiment, the heat of step 4 is between 35 and 41 degrees Celsius. This temperature ensures a fast curing of the tissue graft, while in the mould, without destroying or spoiling the biological material. Furthermore, the heating for at least one minute ensures proper integration and adhesion of the biological material to the intermediate sheet and / or the ECM.

[0146] In an advantageous embodiment, the biological graft is a graft for a transplant. It is to be understood, that the biological graft is preferably an autologous tissue graft.

[0147] In yet another preferred embodiment, the extracellular matrix is a solution comprising at least one of collagen, fibronectin, platelet rich plasma, or gelatine.

[0148] The extracellular matrix material may further be selected from one or more of the following:

[0149] • Elastin

[0150] • Laminin

[0151] • Hyaluronic Acid

[0152] • Plasma serum hylanuroic acid

[0153] • Chitosan

[0154] • Agarose

[0155] • Alginate

[0156] • Matrigel

[0157] • Polyethylene Glycol (PEG)

[0158] • Poly(lactic-co-glycolic acid) (PLGA)

[0159] It is further to be understood, that any of the ECM materials may be tailored to specific needs of a patient receiving the tissue graft.

[0160] In a preferred embodiment, the layer of extracellular matrix, typically fully covering the biological material, is between 2 and 25 mm, preferably between 4 and 25 mm even more preferably between 8 and 20 mm, prior to step 4. It is to be understood, that should the layer of ECM be thicker than e.g. 18 mm, hl of the receptacle would be higher than e.g. 18 mm, such as at least 20 mm. Thus, it is 85186PC01

[0161] 17 to be understood, that the receptacle may be formed to accommodate more or less material, depending on the use of the tissue graft to be moulded.

[0162] In another preferred embodiment, the heating of step 4 is applied for between 1 and 10 minutes. Current studies have demonstrated that approximately 10 minutes of heat for curing, of approximately 37 degrees Celsius provides for a structural integrity of the tissue graft, which is easy to work with during the transplant process. It is further to be understood, that heating is a means of curing the layers of graft, and said heating may be replaced with a different curing step, such as light curing with e.g. blue light or other suitable methods.

[0163] In yet another preferred embodiment, the ECM is mixed with a buffer solution, prior to performing step 2. Such a buffer may e.g. be used to ensure that the pH is in a required range and / or it may be used to add nutrients, such as sugar, to the cells of the biological material in order to improve survival of the cells of the tissue graft until it can later receive nutrients from the body of the patient after the transplantation.

[0164] In an advantageous embodiment, the biological graft, after step 6, is between 0.5 and 5 mm, preferably between 1 and 3 mm, thick.

[0165] In another advantageous embodiment, the biological material is minced prior to step 1. By mincing the biological material, a smaller autologous tissue biopsy is needed, and thereby a smaller wound at the site of harvesting. Yet, a large defect can be covered by the autologous tissue graft by spreading out the minced biological particles.

[0166] It is further to be understood, that the autologous tissue will biologically expand after transplantation, i.e., to cover a larger area than its initial size.

[0167] In yet other embodiments, the ECM or buffer may be replaced by, or further comprising a further component, enabling the tissue graft to cure at ambient temperature, as is known from other 2-component curing materials, such as 2k glue. It is to be understood, that 2k glue is not suitable for tissue grafts. 85186PC01

[0168] 18

[0169] In a third aspect, the invention relates to a kit of parts for preparing a tissue graft from biological material, the kit comprising the mould according to the first aspect and an extracellular matrix material, ECM.

[0170] This embodiment is particularly advantageous for providing means to health professionals, such as surgeons, to rapidly mould an autologous tissue graft to a patient, in need of such a tissue graft. It is to be understood, that such a tissue graft may be moulded in less than 30 minutes, when using the mould according to the first aspect of the invention and performing the method according to the second aspect of the invention.

[0171] In a preferred embodiment, the kit of parts further comprises a buffer solution.

[0172] In another preferred embodiment, the kit of parts further comprises a mixing receptacle or mixing device adapted for mixing the ECM material with a buffer solution.

[0173] In yet another preferred embodiment, the kit of parts further comprises a mincing device adapted to mince biological material. Such a mincing device may e.g. be a scalpel.

[0174] In yet another preferred embodiment, the kit of parts further comprises a sterile transportation box for transporting the mould to a heating cabinet.

[0175] In a preferred embodiment, the kit of parts further comprises a sterile packaging for at least the mould.

[0176] In another preferred embodiment, the kit of parts further comprises instructions for use, detailing the steps of the second aspect of the invention.

[0177] In a fourth aspect, the invention relates to the use of a mould according to the first aspect, for the provision or manufacturing of a tissue graft, such as a tissue graft for an animal or a human. 85186PC01

[0178] 19

[0179] In a fifth aspect, the invention relates to a tissue graft manufactured by the use of the mould according the first aspect or the method according to the second aspect.

[0180] In a sixth aspect, the invention relates to the treatment of a patient, by use of a tissue graft manufactured by using the mould according the first aspect or the method according to the second aspect.

[0181] The first, second, third, fourth, fifth and sixth aspect of the present invention may each be combined with any of the other aspects. These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

[0182] BRIEF DESCRIPTION OF THE FIGURES

[0183] The mould according to the invention will now be described in more detail with regard to the accompanying figures. The figures show one way of implementing the present invention and is not to be construed as being limiting to other possible embodiments falling within the scope of the attached claim set.

[0184] FIG. 1 shows a trimetric top view of a mould, according to an embodiment of the invention;

[0185] FIG. 2 shows a top view of the mould in FIG.l;

[0186] FIG. 3 shows a side view of the mould in FIG. 1;

[0187] FIG. 4 shows a trimetric bottom view of the mould in FIG. 1;

[0188] FIG. 5 shows a trimetric top view of an illustration of a mould, according to another embodiment of the invention;

[0189] FIG. 6 shows a trimetric top view of an illustration of a mould, according to yet another embodiment of the invention;

[0190] FIG. 7 shows a trimetric view of an illustration of a mould, according to yet another embodiment of the invention;

[0191] FIG. 8 shows a trimetric view of an illustration of a mould, according to yet another embodiment of the invention;

[0192] FIG. 9 shows a trimetric view of an illustration of a mould, according to yet another embodiment of the invention; 85186PC01

[0193] 20

[0194] FIG. 10 shows a trimetric view of an illustration of a mould, according to yet another embodiment of the invention;

[0195] FIG. 11 shows a trimetric view of an illustration of a mould, according to yet another embodiment of the invention;

[0196] FIG. 12 shows a trimetric view of an illustration of a mould, according to yet another embodiment of the invention;

[0197] FIG.13 is a flow chart of a method of preparing a biological graft, according to an embodiment of the invention.

[0198] FIG. 14 is a photo of an embodiment of a mould according to the invention. FIG. 15 is a photo of an example of the process of arranging minced biological material on an intermediate sheet in the form of a mesh, before it is moved to the receptacle.

[0199] DETAILED DESCRIPTION OF AN EMBODIMENT

[0200] FIG. 1 shows a trimetric top view of a mould 1, according to an embodiment of the invention. In FIG. 1 the mould 1 comprises a receptacle 10, the receptacle having a bottom surface 11 and sidewalls 12 comprising collapsible portions 13, 13'. It can be seen that the receptacle 10 defines a container suitable for containing the layers of biological material for manufacturing a tissue graft (not shown). It is further shown that the mould 1, when the lid 20 is folded onto the receptacle 10, defines a closed compartment. The receptacle 10 further comprises a hinge 30, the hinge connected to the lid 20. The lid 20 comprises a plurality of through-holes 22. The lid 20 further comprises supports 21, 21' extending substantially perpendicular to a plane of the lid 20. It is to be understood, that the supports 21, 21' are adapted for creating a gap between an associated surface, such as a table, and the lid 20, when the mould 1 is closed, the receptacle 10 being on top of the lid 20, to allow excess fluid draining downwards, through the through-holes 22.

[0201] FIG. 2 shows a top view of the mould 1 in FIG. 1. As seen in FIG. 2 the lid 20 further comprises a locking mechanism 23, adapted to fixate the lid 20 to the receptacle 10, when folded. 85186PC01

[0202] 21

[0203] FIG. 3 shows a side view of the mould 1 in FIG. 1, and FIG. 4 shows a trimetric bottom view of the mould 1 in FIG. 1.

[0204] FIG. 5 shows a trimetric top view of an illustration of a mould 1 according to another embodiment of the invention. In FIG. 5 the mould 1 comprises a receptacle 10, the receptacle having a bottom surface 11, sidewalls 12 and a handle 14. The receptacle 10 further comprises a hinge 30, the hinge connected to a lid 20. The lid 20 comprises a plurality of through-holes 22. The arrow A indicates the folding of the mould 1, after positioning layers of biological material for manufacturing a tissue graft (not shown) within the receptacle 10.

[0205] FIG. 6 shows a trimetric top view of an illustration of a mould 1, according to yet another embodiment of the invention. In FIG. 6 the mould 1 comprises a receptacle 10, the receptacle having a bottom surface 11, sidewalls 12 and a locking mechanism 23. The receptacle 10 further comprises a hinge 30, the hinge connected to a lid 20. The lid 20 comprises a plurality of through-holes 22. The lid 20 further comprises supports 21, 21' extending substantially perpendicular to a plane of the lid 20. The arrow A indicates the folding of the mould 1, after positioning layers of biological material for manufacturing a tissue graft (not shown) within the receptacle 10.

[0206] FIG. 7 shows a trimetric view of an illustration of a mould 1, according to yet another embodiment of the invention. In FIG. 7 the mould 1 is turned upside down for draining of excess fluid from material the inner compartment. The mould 1 comprises a receptacle 10 connected to a lid 20. The lid 20 comprises a plurality of through-holes (see e.g. FIG. 1 or FIG 2). The lid 20 further comprises supports 21, 21' extending substantially perpendicular to a plane of the lid 20. The mould 1 is forced together by a resilient member RM, RM', RM". The exact shape, elastic properties, and fastening mechanism of the resilient member will be determined as part of the design process. The design process may e.g. comprise computer simulations, physical tests, or a combination thereof.

[0207] FIG. 8 shows a trimetric view of an illustration of a mould 1, according to yet another embodiment of the invention. The mould 1 comprises a receptacle 10 and a lid 20 adapted to be slid from a side, to fixate said lid 20 to the receptable 10. 85186PC01

[0208] 22

[0209] The receptacle 10 comprises a bottom surface 11, collapsible sidewalls 12, and first and second locking members LM, LM' protruding from the receptacle 10 and adapted for receiving the lid 20 from a side of said receptacle 10. It is to be understood, that the lid 20 has corresponding surfaces to engage with the locking members LM, LM'.

[0210] FIG. 9 shows a trimetric view of an illustration of a mould 1, according to yet another embodiment of the invention. The mould 1 comprises a receptacle 10 and a lid 20 inserted within the receptacle 10. The lid comprises first and second resilient members RM, RM' adapted to engage with the locking mechanism 23 of the receptacle 10. It is to be understood, that when the resilient members RM, RM' is engaged with the locking mechanism 23, an elastic force of the resilient members RM, RM' forces the lid 20 into the receptacle 10 and towards the bottom thereby applying a force F to the biological material arranged in the mould during use.

[0211] FIG. 10 shows a trimetric view of an illustration of a mould 1, according to yet another embodiment of the invention. The mould 1 comprises a receptacle 10 adapted to be inserted into the lid 20. The lid 20 comprises a resilient member RM adapted to surround the receptacle 10, when the receptacle 10 is inserted from a side of the lid 20.

[0212] FIG. 11 shows a trimetric view of an illustration of a mould 1, according to yet another embodiment of the invention. The mould 1 comprises a receptacle 10 adapted to be inserted into the lid 20. The lid 20 comprises a resilient member RM in the shape of a spring, the resilient member inserted between the lid 20 and a bracket BR extending from the lid 20. The resilient member RM further comprises a pressure plate PP, and the receptacle 10 is sandwiched between the pressure plate PP and the lid 20. The pressure plate PP is used to distribute the force from the spring over a larger area, but the mass of the pressure plate may also be used to apply a part of the force F.

[0213] FIG. 12 shows a trimetric view of an illustration of a mould 1, according to yet another embodiment of the invention. The mould 1 comprises a receptacle 10 inserted into the lid 20. The lid 20 comprises a resilient member RM, the resilient 85186PC01

[0214] 23 member inserted between the lid 20 and a bracket BR extending from a pressure plate PP, and wherein the receptacle 10 is sandwiched between the pressure plate PP and the lid 20.

[0215] FIG. 13 is a flow chart of a method of preparing a biological graft, according to an embodiment of the invention comprising the use of an intermediary sheet, the method comprising the following steps (SI to S8) :

[0216] 51 -providing the mould according to the first aspect of the invention,

[0217] 52 -layering the biological material by the following steps:

[0218] 53 -providing biological material to the receptacle and / or the intermediary sheet,

[0219] 54 -providing an extracellular matrix material to the receptacle and / or the intermediary sheet,

[0220] 55 -inserting the intermediary sheet within the receptacle; wherein at least one of step 3 and step 4 may be performed in any order and / or repeated prior to or after step 5; and after layering the graft, performing the following further steps:

[0221] 56 -providing heat to the receptacle for at least 30 seconds,

[0222] 57 -positioning the lid on the receptacle, and

[0223] 58 -providing pressure P to the layered biological graft, the pressure typically being between 325 Pa and 660 Pa.

[0224] FIG. 14 is a photo of an embodiment of a mould 1 according to the invention. The receptacle 10 has been filled with ECM. The figure shows how the lid is provided with a plurality of through-going holes 22.

[0225] FIG. 15 is a photo of an example of the process of arranging minced biological material on an intermediate sheet 35 in the form of a mesh, before it is moved to the receptacle 10.

[0226] In short, the present invention relates to a mould 1, the mould 1 comprising a receptacle 10 and a lid 20 for forming a tissue graft, such as an autologous tissue graft for transplant and suturing. The mould 1 is adapted to contain layers of a biological material for manufacturing a tissue graft, providing a specific amount of 85186PC01

[0227] 24 pressure to the biological material and drain the biological material and thereby the tissue graft for excess fluid, thus providing a fast and effective means for building a tissue graft. The mould 1 may comprise one or more resilient members RM, RM', RM", and at least one or more of the resilient members RM, RM'. RM" may be suitable to provide a specified force to apply the desired pressure to the tissue graft. The mould may further be provided with a weighted element, the weighted element adapted with a mass correlating to an area of the receptacle, thus providing a specific amount of force and thus pressure to the mould.

[0228] Although the present invention has been described in connection with the specified embodiments, it should not be construed as being in any way limited to the presented examples. The scope of the present invention is set out by the accompanying claim set. In the context of the claims, the terms "comprising" or "comprises" do not exclude other possible elements or steps. Also, the mentioning of references such as "a" or "an" etc. should not be construed as excluding a plurality. The use of reference signs in the claims with respect to elements indicated in the figures shall also not be construed as limiting the scope of the invention. Furthermore, individual features mentioned in different claims, may possibly be advantageously combined, and the mentioning of these features in different claims does not exclude that a combination of features is not possible and advantageous.

Claims

85186PC0125CLAIMS1. A mould (1) adapted to shape a biological material into a tissue graft, the mould (1) comprising :-a receptacle (10) comprising one or more peripheral sidewall(s) (12) having an initial height hl and a bottom surface (11) having an area al, said sidewall(s) (12) and bottom surface (11) defining an inner compartment for containing the biological material during use of the mould (1) , the one or more peripheral sidewall(s) (12) being adapted to fold or collapse to a final height h2 matching a thickness of the tissue graft, during use of the mould (1), wherein h2 > 0.5 mm and h2 < hl, and-a lid (20) adapted to fit onto the receptacle (10) to close the inner compartment, wherein the lid (20) and the receptacle (10) are manufactured from a biocompatible material; such as a polymer material, a metal material, a cellulose- based material, or a combination thereof, wherein the one or more peripheral sidewall(s) (12) is / are adapted to fold or collapse when a force F is applied to the mould (1) during use, the force F moving the lid (20) and the bottom surface (11) towards each other to create a pressure P, defined by F / al, which pressure is thereby applied to the biological material during shaping of the tissue graft, and wherein the one or more peripheral sidewall(s) (12) are liquid-tight, and the lid (20) and / or any air gap arising between the sidewall(s) (12) and the lid (20), after fitting the lid (20) onto the receptacle (10) during use, are configured so that fluid can be drained from the inner compartment only therethrough.

2. The mould (1) according to claim 1, wherein the mould (1) is adapted to sustain a pressure P of at least between 325 Pa and 660 Pa during use.85186PC01263. The mould (1) according to claim 1 or 2, the lid (20) and the receptacle (10) attached to each other by a hinge (30), the receptacle (10) and lid (20) adapted to fold together thereby defining the closed inner compartment.

4. The mould (1) according to any of the preceding claims, the lid (20) and receptacle (10) made in one piece, such as moulded in one piece.

5. The mould (1) according to any of the preceding claims, wherein the one or more peripheral sidewalls (12) of the mould (1) is / are pleated.

6. The mould (1) according to any of the preceding claims, the mould (1) further comprising a locking mechanism (23) adapted to secure the lid (20) to the receptacle (10) during use of the mould (1).

7. The mould (1) according to any of the preceding claims, the mould (1) further comprising a resilient member (RM, RM', RM") adapted to provide the force F between the lid (20) and the bottom surface (11) of the receptacle (10).

8. The mould (1) according to claim 7 when dependent on claim 6, wherein the resilient member (RM, RM', RM") is comprised in the locking mechanism (23).

9. The mould (1) according to any of the preceding claims, wherein at least a portion of the one or more peripheral sidewall(s) (12) of the receptacle (10) is resilient.

10. The mould (1) according to any of the preceding claims, the lid (20) further comprising at least one through hole (22), such as a plurality of though holes (22).

11. The mould (1) according to any of the preceding claims, wherein the bottom surface (11) is provided with markings, such as grid-shaped markings, configured to visually guide the manual placement of biological material and thereby ensure a predetermined arrangement of the biological material during use of the mould (!)■85186PC012712. Mould (1) according to any of the preceding claims, wherein the mould (1) is provided with one or more protrusions dimensioned and arranged to ensure that the lid (20) and the bottom surface (11) will not be moved closer to each other than to a distance matching the final height h2 of the one or more sidewall(s) (12).

13. The mould (1) according to any of the preceding claims, the mould (1) further comprising an intermediary sheet (35) adapted for insertion between the bottom surface (11) of the receptacle (10) and the lid (20).

14. The mould (1) according to claim 13, the intermediary sheet (35) being a biocompatible mesh.

15. The mould (1) according to any of the preceding claims, wherein hl is between 5 and 30 mm, preferably between 5 and 20 mm.

16. A method of preparing a tissue graft from a biological material, the method comprising the following steps:-providing the mould according to any of claims 1 to 15, -layering the biological material by the following steps:-step 1 : providing biological material to the receptacle and / or the intermediary sheet, when present,-step 2: providing an extracellular matrix material to the receptacle and / or the intermediary sheet, when present,-step 3: when using an intermediary sheet, inserting the intermediary sheet within the receptacle; wherein at least one of step 1 and step 2 may be performed in any order and / or repeated prior to or after step 3; and after layering the biological material, performing the following further steps:-step 4: supplying energy to the mould containing the biological material in order to provide a curing of the biological material, -step 5: positioning the lid on the receptacle, and-step 6: applying a force F to the mould thereby applying pressure P, such as pressure being between 325 Pa and 660 Pa, to the layered biological material to shape it into a tissue graft.85186PC012817. Method according to claim 16, wherein step 4 is performed by providing heat to the receptacle (10) for at least 30 seconds.

18. The method according to claim 16 or 17, the method further comprising the following step, after step 5:-turning the mould (1) upside down, the lid (20) facing downwards, to drain any fluid from within the receptacle (10).

19. A kit of parts for preparing a tissue graft form biological material, the kit comprising the mould (1) according to any of claims 1 to 15 and an extracellular matrix material (ECM).

20. Kit according to claim 19, wherein the kit further comprises one or more of the following : a buffer solution for mixing with the ECM, a mixing receptacle or a mixing device adapted for mixing the ECM with a buffer solution, mincing devices adapted to mince biological material, a sterile transportation box for transporting the mould to a heating cabinet, a sterile packaging for at least the mould, and instructions for use.

Images