Weighted mould and method for tissue graft manufacturing

Abstract

The present invention relates to a mould (1) for forming a tissue graft, preferably an autologous tissue graft for transplantation and suturing. The mould comprises a receptacle (10) and a pressure plate (30). 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 receptacle (30) is adapted with a mass m, the mass adjusted to provide the specific pressure, relative to the area of a bottom surface (11) of the receptacle, wherein the tissue graft is to be manufactured. In some embodiments, 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 biological material arranged in the mould during use.

Description

[0001] 85187PC01

[0002] 1

[0003] WEIGHTED 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. 85187PC01

[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 sidewalls having a height hl and a bottom surface having an area al, the sidewall(s) and bottom surface defining an inner compartment for containing the biological material during use of the mould, the receptacle having a mass m, and

[0018] -a pressure plate comprising a graft surface and a top surface, the graft surface defining an area a2, wherein a2 < al, wherein the pressure plate is adapted to extend into the inner compartment until a height h2 from the bottom surface, wherein h2 < hl, wherein, during use of the mould, when the receptacle is arranged on top of the pressure plate with the bottom surface facing downwards, so that biological material is thereby located between the upwards-facing graft surface and the bottom surface, the receptacle applies a pressure P to the biological material thereby shaping the tissue graft, the pressure being defined by P = (rm 9.81 [m / s2]) / al, and 85187PC01

[0019] 3 wherein the one or more peripheral sidewall(s) are liquid-tight, and the pressure plate and / or any air gap arising between the sidewall(s) and the pressure plate, after extending the pressure plate into the inner compartment during use, are configured so that fluid can be drained from the inner compartment only therethrough.

[0020] The term "extend into" may also be described as "be moved partly into" or "be forced into".

[0021] In some embodiments of the invention, the mass m of the receptacle and the area al of the bottom surface are determined to provide a pressure P between 325 Pa and 660 Pa to the biological material during use of the mould.

[0022] During the development of the present invention, this pressure range was 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 using receptacles with different masses m and analysing resulting tissue crafts 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.

[0023] In a more preferred embodiment, the pressure is between 400 and 600 Pa.

[0024] 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 and used to prepare the tissue graft.

[0025] In the context of the present invention, it is to be understood, that the pressure plate is adapted to be forced into the receptacle, until a height h2, and wherein h2 is defined as a gap between the graft surface of the pressure plate and the bottom 85187PC01

[0026] 4 surface of the receptacle, thus, ensuring that the tissue graft is not spoiled or destroyed by the gap closing.

[0027] The term "pressure plate" is used about the part of the mould that carries the biological material during the application of pressure. Thus, it is not this part of the mould that applies the pressure.

[0028] The term "bottom surface" refers to the orientation of the mould, when the biological material is being arranged in the inner compartment, during use of the mould. When the mould is closed and subsequently turned upside down in order to apply the pressure to the biological material by the mass of the receptacle, this "bottom surface" becomes located at the upper region of the mould and facing downwards towards the biological material. Correspondingly, the term "top surface" refers to the upper surface of the pressure plate, before the mould is turned upside down.

[0029] In other words, the mould is adapted to as to provide a gap between the bottom surface of the receptacle and the graft surface of the pressure plate, when pressure is applied and the tissue graft is drained of excess fluid by controlled compression. 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.

[0030] In the context of the present invention, it is to be understood, that the mould must sustain at least in between 325 and 660 Pa, without the pressure plate, the sidewalls or the bottom surface buckling, bulging or coming apart during pressure application.

[0031] 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 curing process, such as a heating process which is used for curing the layers of the graft as will be described below.

[0032] Even further, it is to be understood, that when an autologous tissue graft is being prepared, a heating process is typically applied, and thus the mould is adapted to 85187PC01

[0033] 5 be stable during said heating process, i.e. the mould must be stable at least between 20 and 41 degrees Celsius which is a typically used temperature range.

[0034] It is to be understood, that the heating process is a means of curing the layers of the graft, prior to applying pressure to said graft.

[0035] 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 pressure plate is to be made in a shape corresponding to the shape of the bottom surface.

[0036] 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, that any shape fitting for the tissue graft position, on or in a patient, may be provided by the mould.

[0037] The tissue graft could also be used for in vitro tissue culturing aiming at research studies and pharmaceutical testing in a 3D culture system.

[0038] In another preferred embodiment, the pressure plate further comprises at least one through hole, such as a plurality of through holes, extending between the graft surface and the top surface to provide at least one canal between the bottom surface of the receptacle and an outside environment, after the mould has been closed during use. Such holes are used for draining fluid from the biological material, thereby obtaining a tissue graft with the required properties.

[0039] 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 under-pressure 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. 85187PC01

[0040] 6

[0041] 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. The space between the mould and the tray forms part of the outside environment.

[0042] The above embodiments are particularly advantageous for ensuring correct draining of excess fluids, during the application of pressure.

[0043] In some embodiments of the invention, the bottom surface 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.

[0044] In some embodiments of the invention, the mould is provided with one or more protrusions dimensioned and arranged to ensure that the graft surface and the bottom surface will not be moved closer than to a distance matching the height h2.

[0045] 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 functioning as stops makes it easier to ensure that the desired thickness of the tissue graft is obtained.

[0046] In an advantageous embodiment, the mould further comprises an intermediary sheet adapted for insertion between the bottom surface of the receptacle and the graft surface of the pressure plate.

[0047] In another advantageous embodiment, the intermediary sheet has an area a3, wherein a3 < al.

[0048] In yet another advantageous embodiment, the intermediary sheet is a biocompatible mesh. It may e.g. be polyester-based as is typically used for 85187PC01

[0049] 7 absorbable sutures and surgical meshes. Such a kind of mesh will be known on its own by a person working within this field.

[0050] In a preferred embodiment, the intermediary sheet has a thickness of between 0.01 and 3 mm.

[0051] It is to be understood, that the intermediary sheet is provided as a structural layer, such as a scaffold, and wherein biological material and extracellular matrix (ECM) is absorbed, fused or intertwines with the intermediary sheet, thus providing for a structurally stable tissue graft, suitable for transplant. 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.

[0052] The intermediate sheet may further be selected from acellular auto- / allo- / or xenogenic biological matrices, such as acellular small intestinal submucosa, acellular dermis or acellular bladder matrix.

[0053] 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.

[0054] In another preferred embodiment, the pressure plate further comprises one or more side surfaces; the side surface(s) extending between said graft surface and top surface and / or further extending perpendicular from the top surface. This embodiment is particularly advantageous for ensuring that the pressure plate can be removed from the receptacle, when the tissue graft is prepared. Further, the side surfaces may extend outside the receptacle. This embodiment may be particularly advantageous for providing a support, the support enabling the pressure plate to rest on a surface, such as a table, and wherein the mass of the receptacle, when the receptacle is oriented over the pressure plate, slides downwards, through gravity, exerting a specific pressure on the tissue graft. An example of such an embodiment will be shown in the figures. 85187PC01

[0055] 8

[0056] In yet another preferred embodiment, the one or more side surfaces of the pressure plate have a height h3 and h3 < hl. It is to be understood, that the one or more side surfaces, when adapted to extend outside the receptacle and function as a support for the mould, h3 may be greater or equal to hl, but wherein the graft surface of the pressure plate is not enabled to abut the bottom surface of the receptacle, either by a provided stop, such as protrusions extending radially outwards from the one or more side surfaces, abutting a rim of the receptacle, or by the one or more sidewalls of the receptacle being radially inwards inclining from the rim to the bottom surface.

[0057] In an advantageous embodiment, portions of the one or more side surfaces of the pressure plate comprise recesses, the recesses extending radially inwards, towards the graft surface. This embodiment may be advantageous to prevent the pressure plate from bottoming out, destroying the graft during the application of pressure.

[0058] In another advantageous embodiment, portions of the one or more sidewalls of the receptacle comprise recesses, the recesses extending radially inwards, towards the bottom surface.

[0059] The above embodiments are particularly advantageous for guiding the pressure plate into the receptacle, ensuring correct positioning of the pressure plate, and furthermore serves to prevent sideways movement of the pressure plate, relative to the receptacle, during the application of pressure.

[0060] In yet another advantageous embodiment, the top surface of the pressure plate defines an area a4, and a4 > al.

[0061] In a preferred embodiment, the top surface of the pressure plate extends radially outwards, perpendicular to the one or more side surfaces of the pressure plate, defining a lip.

[0062] The above embodiments are particularly advantageous to provide a stop, wherein the top surface or lip is dimensioned so as to abut a rim of the receptacle, thus 85187PC01

[0063] 9 ensuring that h2 cannot be zero, i.e. ensuring a gap between the graft surface of the pressure plate and the bottom surface of the receptacle.

[0064] In another preferred embodiment, the lip comprises a sealing element adapted to seal between an upper surface of the receptacle and the lip of the pressure plate. This embodiment may be particularly advantageous for providing correct drainage of excess fluid from the tissue graft, wherein through holes of the pressure plate define a specific amount of drainage over time.

[0065] In yet another preferred embodiment, at least one of the receptacle or the pressure plate is made from a metal.

[0066] The metal may preferably be selected from one or more of the following: Titanium and its alloys, stainless steel, tantalum, cobalt-chromium alloys, nickel-titanium (Nitinol), magnesium alloys, zirconium, gold, silver, and platinum.

[0067] In other preferred embodiments, at least one of the receptacles or the pressure plate is manufactured from a polymer. Examples of possible polymers are: 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).

[0068] It is to be understood, that at least the receptacle is required to have a certain mass, in order for gravity to provide the force required to generate sufficient pressure within the mould during the preparation of the tissue graft.

[0069] In yet other preferred embodiments, the receptacle comprises a weighted element removeable from said receptacle, the weighted element comprising between 40 and 99 percent, preferably between 70 and 98 percent, of the mass m. This embodiment is particularly advantageous for enabling the receptacle to be manufactured from a polymer, as the mass necessary to create pressure may be applied from said weighted element then typically being a material with higher density, such as a metal. 85187PC01

[0070] 10

[0071] In most preferred embodiments, the receptacle is manufactured from a metal and the mass m, is integrated within the receptacle. This embodiment provides for accurate / precise tuning of the mass m of the receptacle, relative to the area of the bottom surface of the receptacle, thus providing accurate pressure to the tissue graft, during compression of the tissue graft, after curing.

[0072] In an advantageous embodiment, h2 > 0.5 mm. This embodiment is particularly advantageous to obtain that a gap between the graft surface of the pressure plate and the bottom surface of the receptacle is ensured.

[0073] In another advantageous embodiment, hl is between 5 and 35 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 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.

[0074] In yet another preferred embodiment, at least a portion of the pressure plate extends outside the inner compartment of the receptacle, when the graft surface of the pressure plate is positioned completely inside the inner compartment of the receptacle during use of the mould. This embodiment is particularly advantageous for providing a support, on which the mould can rest, while pressure is applied, ensuring that the receptacle can slide freely downwards through gravity, when the mould is turned upside down, i.e. when the receptacle is positioned on top of the pressure plate, the bottom surface of the receptacle facing downwards.

[0075] In a preferred embodiment, one or more inner surfaces of the mould is / are coated with a biocompatible or bioactive material.

[0076] The biocompatible coating may be selected from one or more of the following :

[0077] • Polycaprolactone (PCL) 85187PC01

[0078] 11

[0079] • Polylactic-co-glycolic acid (PLGA)

[0080] • Polyethylene glycol (PEG)

[0081] • Hydroxyapatite

[0082] • Chitosan

[0083] • Collagen

[0084] • Gelatin

[0085] • Hyaluronic acid

[0086] • Poly(D,L-lactic acid) (PDLLA)

[0087] • Polyvinyl alcohol (PVA)

[0088] The bioactive coating may be selected from one or more of the following:

[0089] • Hyaluronic acid

[0090] • Chitosan

[0091] • Collagen

[0092] • Gelatin

[0093] • Hydroxyapatite

[0094] • Bioactive glass

[0095] • Calcium phosphate

[0096] • Alginate

[0097] • Fibrin

[0098] • Silk fibroin

[0099] It is further to be understood, that one or more of the biocompatible and bioactive coatings may be combined.

[0100] 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.

[0101] In a second aspect, the invention relates to a method of preparing a tissue graft from a biological material, the method comprising the following steps: 85187PC01

[0102] 12

[0103] -providing the mould according to the first aspect of the invention, -layering the biological material by the following steps:

[0104] -step 1 : providing biological material to the receptacle and / or the intermediary sheet, when present,

[0105] -step 2: providing an extracellular matrix material to the receptacle or the intermediary sheet, when present,

[0106] -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:

[0107] -step 4: supplying energy to the mould containing the biological material in order to provide a curing of the biological material,

[0108] -step 5: positioning the pressure plate partly within the receptacle, and -step 6: turning the mould upside down so that the receptacle is arranged on top of the pressure plate, with the graft surface of the pressure plate facing upwards, thereby providing pressure P, such as a pressure between 325 and 660 Pa, to the layered biological material by the mass of the receptacle in order to shape the biological material into a tissue graft.

[0109] 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.

[0110] 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.

[0111] 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, a process which is known from other 2 component curing materials, 85187PC01

[0112] 13 such as 2k glue. It is to be understood, that 2k glue is not suitable for tissue grafts.

[0113] 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.

[0114] 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 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.

[0115] 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.

[0116] It is further to be understood, that the step of turning the mould upside down causes the receptacle to slide downwards, towards the pressure plate, with a specific pressure.

[0117] In a preferred embodiment, step 5 may be performed prior to step 4. 85187PC01

[0118] 14

[0119] In 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.

[0120] In yet another preferred 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.

[0121] 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.

[0122] In another advantageous embodiment, the extracellular matrix ECM is a solution comprising at least one of collagen, fibronectin, platelet rich plasma, or gelatine.

[0123] The extracellular matrix (ECM) material may further be selected from one or more of the following:

[0124] • Elastin

[0125] • Laminin

[0126] • Hyaluronic Acid

[0127] • Plasma serum hylanuroic acid

[0128] • Chitosan

[0129] • Agarose

[0130] • Alginate

[0131] • Matrigel

[0132] • Polyethylene Glycol (PEG)

[0133] • Poly(lactic-co-glycolic acid) (PLGA)

[0134] It is further to be understood, that any of the ECM materials may be tailored to specific needs of a patient receiving the biological graft. 85187PC01

[0135] 15

[0136] In yet another preferred embodiment, the layer of extracellular matrix, at least partly 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 the steps of curing / heating and application of pressure. 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 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. It is further to be understood, that heating is a means of curing the layers of graft, and said heating may be replaces with a different curing step, such as light curing with e.g. blue light or other suitable methods.

[0137] In an advantageous 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.

[0138] In another advantageous embodiment, the biological graft, after step 6, is between 0.5 and 5 mm, preferably between 1 and 3 mm, thick.

[0139] In a preferred embodiment, the heating of step 4 is applied for between 1 and 10 minutes. 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.

[0140] 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 of the invention and an extracellular matrix ECM, material.

[0141] 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 graft. It is to be understood, that such a graft may be 85187PC01

[0142] 16 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.

[0143] In a preferred embodiment, the kit of parts further comprises a buffer solution.

[0144] In another preferred embodiment, the kit of parts further comprises a mixing receptacle or mixing devices adapted for mixing the ECM material with a buffer solution.

[0145] In yet another preferred embodiment, the kit of parts further comprises mincing devices, such as a scalpel and a cutting board, adapted to mince biological material. 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 the minced biological particles.

[0146] In yet another preferred embodiment, the kit of parts further comprises a sterile transportation box for transporting the mould to a heating cabinet.

[0147] 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.

[0148] In an advantageous embodiment, the kit of parts further comprises a sterile packaging for at least the mould.

[0149] In another advantageous embodiment, the kit of parts further comprises instructions for use, detailing the steps of the second aspect of the invention.

[0150] 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. 85187PC01

[0151] 17

[0152] 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.

[0153] In a sixth aspect, the invention relates to the treatment of a patient, by use of a tissue graft manufactured by the use of the mould according the first aspect or the method according to the second aspect.

[0154] 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.

[0155] BRIEF DESCRIPTION OF THE FIGURES

[0156] 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.

[0157] FIG. 1 shows a trimetric view of a mould, according to an embodiment of the invention;

[0158] FIG. 2 shows a trimetric top view of the pressure plate of the mould in FIG. 1;

[0159] FIG. 3 shows a trimetric bottom view of the pressure plate in FIG. 2 turned upside down;

[0160] FIG. 4 shows a side view of the pressure plate in FIGS. 2 and 3;

[0161] FIG. 5 shows a trimetric view of the receptacle of the mould in FIG. 1;

[0162] FIG. 6 shows a side view of the mould in FIG. 1;

[0163] FIG. 7 shows a side view of the mould in FIG. 6 turned upside down;

[0164] FIG. 8 shows a trimetric view of a schematic illustration of the separate parts of the mould, according to an alternative embodiment of the invention;

[0165] FIG. 9 shows a trimetric view of a schematic illustration of the stacked mould of FIG. 8;

[0166] FIG. 10 is a flow-chart of a method of preparing a biological graft according to an embodiment of the invention; 85187PC01

[0167] 18

[0168] FIG. 11 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.

[0169] DETAILED DESCRIPTION OF AN EMBODIMENT

[0170] FIG. 1 shows a trimetric view of a mould 1, according to an embodiment of the invention. The mould 1 comprises a pressure plate 30, inserted partly within an inner compartment of a receptacle 10. Detailed features of the pressure plate will be described in relation to FIG. 2 to FIG. 4. Detailed features of the receptacle 30 will be described in relation to FIG. 5. It is to be understood, that the tissue graft (not visible) is to be build or layered in the receptacle 10, and afterwards moulded in between the receptacle 10 and the pressure plate 30. It is further to be understood, that the receptacle 10 comprises a mass m, the mass m specified to ensure a correct amount of pressure in between the receptacle 10 and the pressure plate 30. Thus, for the pressure to be exerted, the mould 1 is to be turned upside down (see FIG. 7), relative to FIG. 1, enabling excess fluid from the tissue graft to drain through the pressure plate 30, while gravity pulls the receptacle 10 towards the pressure plate 30, thereby moulding / forming the tissue graft during the step of applying pressure.

[0171] FIG. 2 shows a trimetric top view of the pressure plate 30 of the mould in FIG. 1. The pressure plate 30 comprises side surfaces 32 extending perpendicular from a main plane of said pressure plate 30. The side surfaces 32 furthermore extends radially outwards, defining a lip 33. The receptacle 30 further comprises a guide 34, the guide 34 adapted to engage with correlating recesses of a receptacle (see FIG. 5). It is to be understood, that the guide 34 ensures easy positioning of the pressure plate 30 into the receptacle, preventing shear forces to be applied to the surfaces of a tissue graft, between a graft surface 31' of the pressure plate 30 and the bottom surface 11 of the receptacle 10. The pressure plate 30 further comprises a plurality of through holes 36, extending between the top surface 31 and the graft surface 31' (see FIG. 3) of the pressure plate 30. The lip 33 of the side surface 32 further extends downwards, creating stops 35, 35', preventing the pressure plate 30 from bottoming out, into the receptacle, by abutting a rim of the receptacle (see FIG. 7), to prevent excess compaction of the tissue graft and 85187PC01

[0172] 19 to enable fluid to drain. It is to be understood, that the stops 35, 35' create a gap, h2, which defines an end thickness of the moulded tissue graft.

[0173] FIG. 3 shows a trimetric bottom view of the pressure plate 30 in FIG. 2 when turned upside down compared to the orientation in FIG. 2. In this figure, the lip 33 of the side surface 32 further extends upwards. It is further to be understood, that the pressure plate 30 will be oriented in this manner, i.e. upside down, during the moulding process, wherein pressure will be applied to the tissue graft. From FIG. 3 it can further be seen that the lip 33 of the side surfaces 32 has recesses 37, 37'. The guides 34, 34' further have recesses 38. It is to be understood, that the recesses 37, 37', 38 provides for easier insertion of the pressure plate 30 into the receptacle.

[0174] It is to be understood, that the recesses 37, 37' and 38 may also be denoted chamfers.

[0175] FIG. 4 shows a side view of a pressure plate 30 of FIGS. 2 and 3. From FIG. 4 it can further be seen that the lip 33 of the side surfaces 32 has recesses 37, 37'. The guides 34, 34' further have recesses 38, 38'. It is to be understood, that the recesses 37, 37', 38, 38' provides for easier insertion of the pressure plate 30 into the receptacle.

[0176] FIG. 5 shows a trimetric view of the receptacle 10 of the mould in FIG. 1. The receptacle comprises a bottom surface 11 adapted to receive layers of biological material for forming a tissue graft (not shown). From the bottom surface 11, sidewalls 12 extend around a periphery of the bottom surface 11. The receptacle 10 further comprises a rim 13, the rim adapted with recesses / chamfers 15. It is to be understood, that the recesses / chamfers 15 provides for easier insertion of the pressure plate 10. Further, the receptacle comprises inwards arches 14, 14' the arches adapted to accommodate the guides 34, 34' of the pressure plate 30.

[0177] FIG. 6 shows a side view of the mould 1 of FIG. 1. FIG. 6 shows the pressure plate 30 inserted into the receptacle 10, the guides 34, 34' guiding the pressure plate 30, relative to the receptacle 10. Furthermore, the stops 35, 35' can be seen, and wherein the stops 35, 35' are adapted to abut the rim 13 of the 85187PC01

[0178] 20 receptacle, prior to the graft surface (see FIG. 2 and FIG. 3) abutting the bottom surface 11 of the receptacle 10. It is to be understood, that after insertion of the pressure plate 30, the mould 1 is turned upside down to initiate the compression process (see FIG. 7).

[0179] FIG. 7 shows a side view of the mould 1 of FIG. 6 when turned upside down compared to the orientation in FIG. 6. FIG. 7 shows the pressure plate 30 inserted completely into the receptacle 10. In FIG. 7 the stops 35, 35' can be seen abutting the rim 13 of the receptacle.

[0180] FIG. 8 shows a trimetric view of a schematic illustration of the separate parts 10, 16, 30 of the mould 1, according to an alternative embodiment of the invention. The mould 1 comprises a receptacle 10, a pressure plate 30 and a weighted element 16. It is to be understood, that in this particular embodiment, the receptacle 10 may be manufactured from a material different from metal, such as a biocompatible polymer, and wherein the weighted element 16 constitutes a substantial part of the mass m, which is necessary to provide the required pressure to mould a tissue graft. It is further to be understood, that the mass m, of the weighted element 16 is correlated to the area of the bottom surface 11 of the receptacle 10.

[0181] FIG. 9 shows a trimetric view of a schematic illustration of the stacked mould 1 of FIG. 8. FIG. 9 shows the stacked parts, i.e. the receptacle 10, the weighted element 16 and the pressure plate 30 shown as separate parts 10, 16, 30 in FIG. 8. The pressure plate 30 is arranged at the bottom, the weighted element 16 is arranged at the top and the receptacle 10 with the cured tissue graft (not shown) is sandwiched in between. It is to be understood, that the pressure plate 30 is adapted to be received into the inner compartment of the receptacle 10, and the weighted element 16, has a mass m, adapted to provide a sufficient force downwards, relative to the area of the bottom surface 11 of the receptacle, as shown in e.g. FIG. 8, thus compressing the tissue graft and removing excess fluids from the tissue graft. 85187PC01

[0182] 21

[0183] FIG. 10 is a flow-chart of a method of preparing a biological graft according to an embodiment of the invention, the method comprising the following steps (SI - S8):

[0184] 51 -providing the mould according to the first aspect of the invention,

[0185] 52 -layering the biological material by the following steps:

[0186] 53 -providing biological material to the receptacle and / or the intermediary sheet,

[0187] 54 -providing an extracellular matrix material to the receptacle and / or the intermediary sheet,

[0188] 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:

[0189] 56 -providing heat to the receptacle for at least 30 seconds,

[0190] 57 -positioning the pressure plate partly within the receptacle, and

[0191] 58 -turning the mould upside down so that the receptacle is arranged on top of the pressure plate, with the graft surface of the pressure plate facing upwards, thereby providing pressure P to the layered biological graft, the pressure possibly being between 325 Pa and 660 Pa.

[0192] FIG. 11 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.

[0193] In short, the present invention relates to a mould 1 for forming a tissue graft, preferably an autologous tissue graft for transplantation and suturing. The mould comprises a receptacle 10 and a pressure plate 30. The mould is adapted to contain layers of a biological material for manufacturing a tissue graft, providing a specific amount of 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 receptacle 30 is adapted with a mass m, the mass adjusted to provide the specific pressure, relative to the area of a bottom surface 11 of the receptacle, wherein the tissue graft is to be made. In some embodiments, the mould may further be provided with a weighted element, the weighted element adapted with a mass correlating to an area of the 85187PC01

[0194] 22 receptacle, thus providing a specific amount of force and thus pressure to the mould.

[0195] 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

85187PC0123CLAIMS1. 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 sidewalls (12) having a height hl and a bottom surface (11) having an area al, the sidewall(s) (12) and bottom surface (11) defining an inner compartment for containing the biological material during use of the mould, the receptacle (10) having a mass m, and- a pressure plate (30) comprising a graft surface (31') and a top surface (31), the graft surface (31') defining an area a2, wherein a2 < al, wherein the pressure plate (30) is adapted to extend into the inner compartment until a height h2 from the bottom surface (11), wherein h2 < hl, wherein, during use of the mould (1), when the receptacle (10) is arranged on top of the pressure plate (30) with the bottom surface (11) facing downwards, so that biological material is thereby located between the upwards-facing graft surface (31') and the bottom surface (11), the receptacle (10) applies a pressure P to the biological material thereby shaping the tissue graft, the pressure being defined by P = (rm 9.81 [m / s2]) / al, and wherein the one or more peripheral sidewall(s) (12) are liquid-tight, and the pressure plate (30) and / or any air gap arising between the sidewall(s) (12) and the pressure plate (30), after extending the pressure plate (30) into the inner compartment 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 mass m of the receptacle (10) and the area al of the bottom surface (11) are determined to provide a pressure P between 325 Pa and 660 Pa to the biological material during use of the mould (!)■3. The mould (1) according to claim 1 or 2, the pressure plate (30) further comprising at least one through hole (36), such as a plurality of through holes85187PC0124(36), extending between the graft surface (31') and the top surface (31) to provide at least one canal between the bottom surface (11) of the receptacle (10) and an outside environment after the mould (10) has been closed during use.

4. 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 (1).

5. 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 graft surface (31') and the bottom surface (11) will not be moved closer than to a distance matching the height h2.

6. 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 graft surface (31') of the pressure plate (30).

7. The mould (1) according to claim 6, the intermediary sheet (35) being a biocompatible mesh.

8. The mould (1) according to any of the preceding claims, the pressure plate (30) further comprising one or more side surfaces (32); the side surface(s) (32) extending between said graft surface (31') and top surface (31) and / or further extending perpendicular from the top surface (31).

9. The mould (1) according to claim 8, wherein the one or more side surfaces (32) of the pressure plate (30) have a height h3 and h3 < hl.

10. The mould (1) according to any of claims 8 or 9, wherein portions of the one or more side surfaces (32) of the pressure plate (30) comprise recesses (37, 37', 38) or chamfers, the recesses or chamfers extending radially inwards, towards the graft surface (31').85187PC012511. The mould (1) according to any of the preceding claims, wherein portions of one or more the sidewalls (12) of the receptacle (10) comprise recesses (15) or chamfers, the recesses (15) or chamfers extending radially inwards, towards the bottom surface (11).

12. The mould (1) according to any of the preceding claims, the receptacle (10) comprising a weighted element (16) removeable from said receptacle (10), the weighted element (16) comprising between 40 and 99 percent, preferably between 70 and 98 percent, of the mass m.

13. The mould (1) according to any of the preceding claims, wherein h2 > 0.5 mm.

14. The mould (1) according to any of the preceding claims, wherein hl is between 5 and 35 mm, preferably between 5 and 20 mm.

15. The mould (1) according to any of the preceding claims, wherein at least a portion of the pressure plate (30) extends outside the inner compartment of the receptacle (10), when the graft surface (31') of the pressure plate (30) is positioned completely inside the inner compartment of the receptacle (10) during use of the mould (1).

16. A method of preparing a tissue graft from a biological material, the method comprising the following steps:-providing the mould (1) according to any of claims 1 to 15, -layering the biological material by the following steps:-step 1 : providing biological material to the receptacle (10) and / or the intermediary sheet (35), when present,-step 2: providing an extracellular matrix material (ECM) to the receptacle (10) and / or the intermediary sheet (35), when present, -step 3: when using an intermediate sheet (35), inserting the intermediary sheet (35) within the receptacle (10); 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; and85187PC0126 after layering the biological material, performing the following further steps: -step 4: supplying energy to the mould (1) containing the biological material in order to provide a curing of the biological material, -step 5: positioning the pressure plate (30) partly within the receptacle (10), and-step 6: turning the mould (1) upside down so that the receptacle (10) is arranged on top of the pressure plate (30), with the graft surface (31') of the pressure plate (30) facing upwards, thereby providing pressure P, such as a pressure between 325 Pa and 660 Pa, to the layered biological material by the mass of the receptacle (10) in order to shape the biological material into a tissue graft.

17. 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 pressure being provided for between 1 minute and 20 minutes.

19. A kit of parts for preparing a tissue graft from 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, the kit further comprising one or more of the following: a buffer solution for mixing with the ECM, a mixing receptacle or mixing devices 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.

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