WO2024094890A1

WO2024094890A1 - An implant for cartilage and meniscus repair

Info

Publication number: WO2024094890A1
Application number: PCT/EP2023/080806
Authority: WO
Inventors: Daniel Kelly; Bin Wang; Xavier BARCELLO GALLOSTRA; Soraya SALINAS FERNANDEZ; Conor Buckley; Orchid GARCIA
Original assignee: College of the Holy and Undivided Trinity of Queen Elizabeth near Dublin
Current assignee: College of the Holy and Undivided Trinity of Queen Elizabeth near Dublin
Priority date: 2022-11-04
Filing date: 2023-11-06
Publication date: 2024-05-10
Anticipated expiration: 2025-05-04
Also published as: EP4611828A1

Abstract

An implant for cartilage and meniscus repair and formed from decellularized cartilage extracellular matrix (dECM) ink is described. The dECM ink has a dECM concentration of 5 to 10% (w/v), preferably a pH of 10-12, and the dECM in the implant is chemically crosslinked. The implant may be fibrous and formed by 3-D printing fibres with the dECM ink to form a fibrous 3-D construct, or it may be formed by casting the dECM ink in a mould. The dECM may include 50 to 350 mM salt. Implants made of tissue-specific dECM can recapitulate the inherent microenvironmental niche of cells, promoting a tissue-specific phenotype. Freeze-drying the implant and then crosslinking the freeze-dried implant has been found to provide an implant with improved mechanical properties.

Description

TITLE

AN IMPLANT FOR CARTILAGE AND MENISCUS REPAIR

Field of the Invention

The present invention relates to an implant for cartilage and meniscus repair. In particular, the invention relates to an implant for repair of soft tissues in high-load synovial joints such as the meniscus of the knee joint.

Background to the Invention

Different decellularisation methods for tissues such as articular cartilage and meniscus have been reported in previous inventions and in the literature, however the mechanical properties of the biomaterials prepared from such ECM projects are poor. In addition, the printability of a hydrogel or ink prepared from such extracellular matrix (ECM) products is either poor (e.g. lack of fidelity or resolution) or not reported. Implants derived from ECM components and/or printed using dECM inks have been soft and fragile with poor mechanical properties (e.g. the commercial Collagen Meniscus Implant (CMI®)), making them unsuitable for immediate load bearing applications. The problem is commonly addressed by mixing the decellularized ECM (dECM) ink with a secondary hydrogel, producing a less biomimetic environment. US4880429A discloses a prosthetic meniscus comprising a porous dry volume matrix of type I collagen fibers interspersed with glycosaminoglycan molecules, wherein said matrix has a substantially wedge shape including a wide central region between two narrow distal tip regions, wherein said collagen fibers are present at a concentration of about 65%-98% by dry weight. Typically such porous scaffolds are relatively soft compared to native meniscal tissue. US4880429A does not disclose how to produce meniscal implants with biomimetic mechanical properties. Nor does it teach how 3D printing techniques can be used to produce implants with anisotropic structure and mechanics mimetic of the native meniscus.

CN111228574A describes a tissue engineered meniscus scaffold formed from a bioink comprising (a) hydrogel material (e.g. HA, alginate etc), (b) cells and (c) and a matrix material which could be dECM, and printing the bioink to form the scaffold. The concentration of the matrix dECM is 0.5 to 2% (w/v). This patents address the poor printability of ECM by diluting it with a second hydrogel of gelatin or HA.

KR20180049712A describes a cell containing bioink for 3-D wet printing. The bioink may consist of dECM, or dECM combined with a water-soluble polymer. The concentration of the dECM is mentioned as being 1-5% and preferably 2-3%. The method describes neutralising the bioink prior to addition of cells. The construct is printed into a coagulation bath. In the example provided, the bioink contains 3% collagen in addition to dECM, and the dECM/collagen mixture is printed into a coagulation bath at neutral pH.

CN112295015A describes a dECM bioink that comprises in addition to dECM an additional material such as polylactic acid or gelatin to the dECM to make the bioink.

KR20180125776A describes a dECM bioink that formed by forming a first hydrogel with a suitable polymer, forming a sol, and then adding dECM to the sol to make the bioink.

In addition, existing meniscus implants fail to recapitulate the native collagenous fibril distribution, alignment and orientation of the native meniscus, which determines its mechanical anisotropy.

There is a clear need for implants with a composition and mechanical properties more closely resembling that of native meniscus tissue.

It is an object of the invention to overcome at least one of the above-referenced problems.

Summary of the Invention

The Applicant has discovered that dECM hydrogels and inks having a high concentration of dECM (e.g. 5-10% w/v) and optionally a high pH (e.g. >10) are suitable for forming mechanically functional implants for cartilage and meniscus repair by casting or 3-D printing without the need for a second hydrogel. The dECM inks and hydrogels of the invention have been used for high resolution printing of large acellular implants (Figure 3) and casting of anatomically shaped meniscal implants into pre-formed moulds (Figure 4. Such dECM derived implants can be chemically crosslinked (e.g. using glutaraldehyde) during or after the fabrication process to improve the mechanical properties. By selecting the appropriate pH, nozzle diameter, temperature and printing speed during the 3D printing of the dECM inks of the invention, the extent of collagen fiber alignment in the resulting print can be controlled (Figures 5 and 6. Moreover, changing the quantity of NaCI added to the dECM ink also changes the conformation of the fibers on a micro-scale. (Figure 1). The importance of scaffold/implant architecture in directing successful tissue engineering has been widely reported, however 3D printing of anisotropic ECM constructs remains challenging. In this invention, control over the fiber formation and alignment through 3D printing can be used to build scaffolds that recapitulate the native meniscus fiber arrangement. In fact, it has been demonstrated that tensile properties of printed dog bones increase due to the orientation of the deposited fibers (Figure 8). Therefore, by adjusting fiber alignment, a dECM implant can be fabricated with different zones containing different structural and mechanical properties, to provide an environment that would mimic the native one (Figure 9). For constructs that are produced either by casting or 3D printing dECM materials, a more porous implant can be produced by freeze-drying the construct post-fabrication. In this scenario, chemically crosslinking the construct after freeze-drying produces an implant with superior mechanical properties (Figure 8). dECM Ink

In a first aspect, the invention provides a decellularized extracellular matrix (dECM) ink comprising at least 5%, for example 5 to 10%, cartilage dECM and typically having a pH range from 4 to 12.

The term “ink” is used to describe the dECM composition, but it could also be referred to as a slurry or a hydrogel as it generally has a viscosity of a gel or viscous slurry.

In any embodiment, the dECM is cartilage or meniscus dECM.

In any embodiment, the ink is an aqueous ink.

In any embodiment, the dECM ink has a pH of 9 to 12 or 10 to 12.

In any embodiment, the dECM ink has a pH of 10.5 to 11.5, or about 11. In any embodiment, the dECM ink comprises a salt.

In any embodiment, the dECM ink comprises 10 mM to 300 mM salt.

In any embodiment, the dECM ink comprises 100 mM to 200 mM salt.

In any embodiment, the dECM ink comprises 125 mM to 175 mM salt.

In any embodiment, the salt is a chloride salt such as, for example, sodium chloride. Other salts, both chlorides and others, may be employed.

In any embodiment, the dECM ink does not comprise a second hydrogel.

In any embodiment, the dECM ink does not comprise a second hydrogel comprising gelatin, hyaluronic acid, dextran or collagen.

The invention also provides an implant comprising (or formed from) dECM ink of the invention.

In any embodiment, the implant is crosslinked.

In any embodiment, the implant is freeze-dried. This makes the implant more porous and also reduced the water content of the implant making it more suitable for storage. The implant may be snap-frozen prior to freeze-drying. When the implant is freeze-dried, it is generally re-hydrated prior to use.

In any embodiment, the implant is crosslinked after freeze-drying. Surprisingly, performing crosslinking after the freeze drying process results in superior mechanical properties and elasticity in the implant.

In any embodiment, the implant is chemically crosslinked with a chemical crosslinking agent. In any embodiment, the crosslinking agent can be natural and/or synthetic and may be selected from, among others, the following ones: genipin (GNP), riboflavin, tissue transglutaminase 2 (TG2), proanthocyanidin (PA), epigallocatechin gallate (EGCG), dehydrothermal treatment (DHT), glutaraldehyde, N-(3- Dimethyl aminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and EDC with N hydroxysulfosuccinimide (EDC-NHS)

In any embodiment, the implant is selected from a cast and a 3-D printed construct.

In any embodiment, the implant comprises casted dECM ink. In any embodiment, the implant is casted in the shape of a natural cartilage tissue (for example in the shape of a meniscus of the knee, or knee articular cartilage).

In any embodiment, the implant comprises 3-D printed dECM ink. This means that it is formed by 3D printing of a dECM ink. The dECM ink is generally printed into fibres, which provides the implant with a fibrous structure. The fibres may be arranged in a manner to mimic the arrangement of fibres in natural meniscus cartilage (see Figures 9 where the fibres are printed into an arc shape mimicking the arrangement of fibres in the meniscus of the knee).

The implant may comprise fibres having a first configuration and fibres having a second configuration. For example, a first set of fibres may be arranged to extend in a first Direction or orientation (e.g. circumferential fibres) and a second set of fibres may be arranged to extend in a second direction or orientation that is different to the first direction or orientation (e.g. radial fibres). The first and second sets of fibres may be arranged in a lattice.

Thus, in any embodiment, the implant is a fibrous implant, in which at least some of the fibres are formed by 3-D printing of a dECM ink of the invention.

In any embodiment, the implant is a fibrous implant, in which: at least some of the fibres are formed by 3-D printing of a first dECM ink of the invention; and at least some of the fibres are formed by 3-D printing of a second dECM ink of the invention. In any embodiment, the first dECM ink has a first salt concentration and the second dECM ink has a second salt concentration that is different to the first salt concentration. The first salt concentration may be less than 150 mM and the second salt concentration may be greater than 150 mM.

In any embodiment, the first dECM ink has a first dECM concentration and the second dECM ink has a second dECM concentration that is different to the first dECM concentration.

In any embodiment, the first dECM ink has a first salt concentration and a first dECM concentration and the second dECM ink has a second salt concentration that is different to the first salt concentration and a second dECM concentration that is different to the first dECM concentration.

In any embodiment, at least some of the fibres are formed by 3-D printing of a third dECM ink of the invention. The third dECM ink of the invention may have a dECM concentration and/or a salt concentration that is different to first and second dECM and salt concentrations.

The width (e.g. thickness) of the printed fibres may be adjusted by modifying the printing speed and/or the salt concentration of the dECM ink. In any embodiment, the printed fibres have a width of at least 100 pm and up to 1500 pm, for example 100 to 1000 pm, or 300 to 800 pm. The salt concentration of the dECM ink may be varied from 0 mM up to 1 M, for example 1 to 500 mM. The 3-D printing speed may be varied from 1-100 mm/s, for example 1-50, 1-30, 5-30 mm/s.

In any embodiment, the implant is fibrous (e.g. it is formed by 3-D printing of a dECM ink) and exhibits a compressive modulus of greater than 60, 70, 80, 90, 100, 120, 140 or 150 KPa.

Medical Use

The invention also relates to the use of an implant of the invention to repair or replace mammalian tissue, for example articular or connective tissue, for example cartilage especially damaged cartilage, for example articular cartilage or meniscus. The damage may be caused by trauma, disease and/or aging. The damage may comprise a defect in the cartilage or meniscus. The implant may be shaped to match the shape of the defect.

The invention also relates to a method of treating a mammalian subject by implanting an implant according to the invention into the subject. The implant may replace damaged native tissue.

The invention also relates to a method of treating cartilage damage in a subject comprising a step of replacing all or some of the damaged cartilage with an implant according to the invention.

In any embodiment, the damaged cartilage is located at a high load bearing location such as the knee.

Method of Manufacture - Ink

The invention also relates to a method of making a decellularized extracellular matrix (dECM) ink comprising the step of mixing dECM with a solvent to provide an ink having a dECM concentration of at least 5%.

In any embodiment, the method comprises adjusting the pH of the ink to a pH of 4 to 12.

In any embodiment, the method comprises adjusting the pH of the ink to a pH of 9 to 12.

In any embodiment, the method comprises adjusting the pH of the ink to a pH of 10 to 12.

In any embodiment, the dECM has a concentration of 5-10% (w/v).

In any embodiment, the dECM has a pH of about 11.

In any embodiment, the method comprises adding salt to the dECM and solvent to provide an ink having a salt concentration of 1 mM to 1M, 10 mM to 500 mM, 100 mM to 300 mM, 100 mM to 200 mM. In any embodiment, the dECM has a reduced GAG content compared to native ECM tissue.

In any embodiment, the dECM is formed by a process comprising the steps of: size-reducing ECM tissue (e.g. meniscal or cartilage tissue); pretreatment of the size-reduced tissue; digestion with pepsin; dialysis with a buffer; and freeze-drying.

In any embodiment, the pretreatment comprises treating the tissue with a surfactant (e.g. a Triton such as Triton-X-100), and/or treating the tissue with benzonase DNase.

In any embodiment, the pretreatment comprises treatment with an alkali such as NaOH;

In any embodiment, the size-reduced ECM tissue is subjected to one or more freeze-thaw cycles prior to the pre-treatment step.

In any embodiment, the buffer is water or a weak buffer (e.g 0.2% sodium hypochlorite).

In any embodiment, the dECM is formed by a process comprising the steps of: size-reducing ECM tissue (e.g. meniscal or cartilage tissue); subjecting the size-reduced tissue to one or more freeze-thaw cycles; treating the tissue with a surfactant (e.g. a Triton such as Triton-X-100); treating the tissue with benzonase DNase; acid solubilisation in acid with pepsin; dialysis with pure water; and freeze-drying.

In any embodiment, the dECM is formed by a process comprising the steps of: size-reducing ECM tissue (e.g. meniscal or cartilage tissue); pretreatment with an alkali such as NaOH; pepsin digestion; salt precipitation; dialysis with a weak buffer (e.g. 0.2% sodium hypochlorite); and freeze-drying.

Method of Manufacture - Implant

The invention also relates to a method of making an implant comprising providing a decellularized extracellular matrix (dECM) ink, and forming an implant from the dECM ink.

In any embodiment, the dECM ink is a dECM ink of the invention.

In any embodiment, the step of forming an implant from the dECM ink comprises (a) casting the dECM ink in a mould or (b) 3-D printing (e.g. extrusion printing) one or more fibres from the dECM ink, and forming the implant from the one or more 3-D printed fibres. Both steps generally result in thermal gelation of the ink and the resultant implant has the characteristics of natural cartilage or meniscus.

In any embodiment, the method comprises freeze-drying the implant.

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In any embodiment, the method comprises crosslinking the implant. The crosslinking may be chemical crosslinking with a chemical crosslinking agent The crosslinking agent may be glutaraldehyde or any of the examples provided above.

In any embodiment, the method comprises freeze-drying the implant and then cross-linking of the freeze-dried implant. Surprisingly, crosslinking after freeze-drying provides an implant with superior mechanical properties.

In any embodiment, the 3-D printing step comprises printing a first part of the implant using a first dECM ink and printing a second part of the implant using a second dECM ink. The first and second dECM inks may differ in their dECM concentration, pH, salt concentration (e.g. 0 mM up to 1 M or more). They may also differ in their method of manufacture (e.g. one ink may be formed by the Protocol 1 and the other ink may be formed by Protocol 2 as described below.

In any embodiment, the 3-D printing step comprises printing a first part of the implant with a first printing protocol and printing a second part of the implant with a second printing protocol. The printing protocol may differ in terms of printing speed or another parameter selected from nozzle diameter, shape and material, temperature of the cartridge and print bed, printing speed and pressure, specified scaffold geometry and infill pattern.

In any embodiment, the 3-D printing step comprises printing the one or more fibres in a configuration that mimics the arrangement of fibres in natural cartilage (see Figures 4 and 5 where the fibres are printed into an arc shape mimicking the arrangement of fibres in the meniscus of the knee).

The implant may comprise fibres having a first configuration and fibres having a second configuration. For example, a first set of fibres may be printed to extend in a first direction or orientation (e.g. circumferential fibres) and a second set of fibres may be printed to extend in a second direction or orientation that is different to the first direction or orientation (e.g. radial fibres). The first and second sets of fibres may be printed in a lattice.

In any embodiment, the casting step is performed in a mould having a shape of a natural cartilage tissue, for example a medical meniscus or articular cartilage tissue.

In any embodiment the casting step comprises introducing the d-ECM at the appropriate salt concentration (150 mM) and pH (11) into a mould. Then, the cast material undergoes a degassing process, typically by introducing it into a vacuum chamber for 15 minutes. After air bubble removal, the cast material is incubated at elevated temperature, for example 4h at 37 °C before removing it from the mould. Once the embodiment is demoulded, it can be crosslinked

In any embodiment, crosslinking the implant comprises immersing the implant in a crosslinking solution for a suitable period of time, for example at least 3, 6, 12, 18 or 24 house, and generally 3-48 house, 12-36 hours, or about 24 hours.

In any embodiment, the crosslinking solution comprises a chemical crosslinking agent and salt. In any embodiment, the salt is the same salt that is present in the dECM and at the same or similar concentration. In any embodiment, the crosslinking solution is an aqueous solution.

In any embodiment, the concentration of the chemical crosslinking agent is about 0.2% to about 1.0%, about o.3% to about 0.8%, or about 0.5% (v/v). In any embodiment, the concentration of the salt in the crosslinking solution is about IQ- 300 mM, 50-250 mM, 100-200 mM, or about 150 mM.

If needed: For the crosslinking, I prepare a solution of 0.5% GA in DI water with the same salt concentration that the implant has(150mM). Then, I adjust the pH of the solution to pH11. I use 1mL of the crosslinking solution for each mL of dissolved dECM.

Then, I introduce the scaffold inside the solution for 24 hours, at RT. After the incubation, The scaffold rinsed with PBS and washed for 4 days with at least 4 PBS changes.

In any embodiment, the concentration of dECM in the ink is chosen to match the ECM density of the tissue being replaced or repaired. For example, for a medical meniscus implant a dECM ink having a dECM concentration of about 40 - 100mg/ml is employed. In contrast, for hyaline cartilage a dECM ink having a concentration of 30 - 100mg/ml is generally employed. In any embodiment, the concentration of dECM for casting is about 60 to 80 mg/ml, and ideally about 70 mg/ml.

In any embodiment, the method comprises drying the implant. Drying may comprise freeze-drying (optionally in combination with an initial snap-freezing step). The dried implant will be rehydrated in water prior to use.

Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

Brief Description of the Figures

Figure 1 : Mechanical properties of P1 dECM casted constructs, (a-c) Compression modulus, equilibrium modulus and dynamic modulus of dECM constructs at 150mM of NaCI and different pHs. (d-f) Compression modulus, equilibrium modulus and dynamic modulus of dECM constructs at pH 11 and different NaCL concentrations.

Figure 2: SEM imaging of d-ECM constructs, (a) Pore and (b) fiber formation within the constructs.

Figure 3: 3D printability of dECM inks, (a) Macroscopic images of dECM inks extruded from a 25 gauge needle, (b) Printed pattern using different dECM inks. (C) Post printing spreading ratio (width of filament divided by needle diameter). ****p < .0001. (d) Printed grid structures with P1 pH11 ink. (e) 3D model of a porcine meniscus, (f) Printing path for 3D printing, (g, h) Images of printed meniscus structures using P1 pH11 ink.

Figure 4: Casted d-ECM meniscus. Top and front view of designed human meniscus model and cast dECM meniscus implant.

Figure 5: Schematics of 3D printing dECM inks to achieve alignment of collagen fibers.

Figure 6: Alignment of collagen fibers through 3D printing, (a) Control of the alignment of collagen fibres by increasing the nozzle speed and controlling the NaCI concentrations, at pH 11. (b) Optical images of 3D printed filaments at v=30 mm/s at different NaCI concentrations. Effect of NaCI concentration over fibre alignment is measured through directionality histograms.

Figure 7: Effect of the fibre alignment over the mechanical properties of the dECM ink. Comparison between the tensile properties of 3D printed dog bones, with a vertical alignment of fibres and cast dog bone specimens, without specific arrangement of the fibres. Printing improves the tensile mechanical properties of the construct.

Figure 8: Comparison of pre and post freeze-drying crosslinking of cast dECM in (a) compression and (b) tension tests.

Figure 9: Schematic of the 3D printing of a dECM meniscus implant. The first layers would include circumferential fibres. Consequently, radial fibres would be interspersed within the circumferential ones. Finally, a superficial layer would be deposited or cast containing a random fibrillar network.

Figure 10: Effect of the freeze-drying process over the internal structure of casted and 3D printed d-ECM. Scaffolds were stained with picrosirius red before performing the freeze- drying process (Before FD), after snap freezing with subsequent freeze drying (LN + FD) and performing the freeze-drying process (After FD). Pictures were taken with polarized light (PL) and without it (Non PL). Figure 11 : Effect of the freeze-drying process on the tensile properties of casted and 3D printed vertical fibre-oriented d-ECM at 7% w/v. Comparison between the tensile properties of rehydrated (a) casted and (b) 3D printed (3DP) dog bones previously subjected to a freeze-drying process: Before performing the freeze-drying process (Before FD), after snap freezing with subsequent freeze drying (LN + FD) and performing the freeze-drying process (After FD).

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

Definitions and general preferences

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise, " or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps. As used herein, the term “disease" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, age, poisoning or nutritional deficiencies.

As used herein, the term "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term “therapy”.

Additionally, the terms "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term “prophylaxis".

As used herein, an effective amount or a therapeutically effective amount of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure. Improvement may be observed in biological I molecular markers, clinical or observational improvements. In a preferred embodiment, the methods of the invention are applicable to humans, large racing animals (horses, camels, dogs), and domestic companion animals (cats and dogs). In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include "individual", "animal", "patient' or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human. As used herein, the term “equine” refers to mammals of the family Equidae, which includes horses, donkeys, asses, kiang and zebra.

In this specification, the term “extracellular matrix tissue” or “extracellular matrix” or “ECM” should be understood to mean a collection of extracellular molecules secreted by cells that provides structural and biochemical support to the surrounding cells. The ECM may be obtained from a mammal, for example a human or a non-human mammal, or it may be engineered in-vitro using published techniques, for example Vinardell et al (Vinardell, T., Sheehy, E., Buckley, C.T., Kelly, D.J. A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cells sources. Tissue Engineering Part A, 18(11-12), 1161-1170, 2012) and Buckley et al (Buckley, C.T., Vinardell, T., Kelly, D.J. Oxygen Tension Differentially Regulates the Functional Properties of Cartilaginous Tissues Engineered from Infrapatellar Fat Pad Derived MSCs and Articular Chondrocytes. Osteoarthritis and Cartilage, 18 (10), 1345-1354, 2010). Examples of extracellular matrix for the purpose of the present invention include cartilage ECM, in particular articular cartilage tissue such as meniscus tissue. The cartilage may be porcine cartilage. The cartilage may be micronized, which means means provided in a particulate form, in which the particles of ECM have a mean particle size of less than 200 microns as determined using routine light microscopy. Preferably, the micronised ECM has a mean particle size of less than 150 or 100 microns. Ideally, the micronized ECM has a mean particle size between 20 and 200 microns, 20 and 150 microns, 20 and 100 microns, 20 and 70 microns, 30 and 70 microns, 30 and 60 microns, 40 and 60 microns, and ideally about 50 microns. Methods of micronisation include milling or cryomilling. An example of a cryomilling machine is the RETCH CRYOMILL™. In this specification, the term “decellularized ECM” or “dECM” refers to ECM tissue that is digested, ideally enzymatically digested, to become soluble in an aqueous solvent and in which the cellular content of the material is reduced partially or preferably completely. Suitably solubilising agents will be known to the person skilled in the art, and include enzymes and denaturing agents such as urea. An example of an enzyme that can be used to digest ECM tissue to become soluble is a protease, for example pepsin, or a collagense. Preferably, the solubilised ECM will be a purified collagen with substantial removal of GAG and xenogeneic DNA. Ideally, the solubilised ECM will have greater than 50%, 60%, 70%, 80% or 90% removal of GAG and DNA when compared to native ECM tissue. Methods of decellularizing ECM are described in PCT/EP2015/068855, PCT/CN2020/12374, US 20140023723, US20150344842, Tissue Engineering: Part A, 2008; 14(4) 2008: 505-518, Journal of Biomaterials Applications. 2021; 35(9): 1192-1207, Acta Biomaterialia, 2021 : 128:175-185.

In this specification, the term “dECM ink” refers to an aqueous slurry of dECM that typically has 5% dECM (generally about 5-10% dECM) or more and typically a pH of at least 9, 10 or 11 (generally about 10-12 and ideally about 11). It may also be referred to herein as a dECM slurry or dECM hydrogel. The dECM may also include a salt, for example NaCI (although other salts may be employed). The salt may be included at a concentration of up to 1 M, for example 50 mM to 500 mM, 50 mM to 350 mM, 100 mM to 200 mM, or about 150 mM. In any embodiment, the dECM ink does not include a second hydrogel, e.g. does not include a hydrogel formed a component other than the dECM.

In this specification, the term “implant” refers to a 3-D construct formed from dECM ink that is suitable for implantation into a subject to replace native tissue such as meniscus or native cartilage tissue, in particular native cartilage tissue of a joint such as a high load bearing joint, or soft tissue, especially soft tissue in high load synovial joints. The implant may be formed by casting in a mould or by 3-D printing of fibres which are arranged in a 3- D shape to make up the joint. The fibres in the printed implant may be arranged to mimic the orientation of fibres in natural cartilage. The implant may be shaped to replace natural cartilage tissue such as articular cartilage or a meniscus. Printed implants may include first fibres and second fibres that are compositionally or dimensionally different. For example, the first and second fibres may be formed from dECM that differ in terms of their dECM concentration, pH, degree or type of crosslinking, or salt concentration, which results in fibres that are compositionally or structurally different. First fibres may be printed using a different printing protocol than second fibres (e.g. faster printing speed) which result in fibres with different thickness. Thus, varying the dECM composition, crosslinking and printing protocols can vary the characteristics of fibres, and allowing large tissue sized meniscus implants to be fabricated that mimic the native tissue (both in terms of the micro and macro structure and mechanical properties). The implant may be freeze-dried. The implant may be crosslinked (e.g. chemically crosslinked). The implant may be freeze-dried and then crosslinked.

In this specification, the term “cross-linked” as applied to the implant or dECM should be understood to mean treated to introduce cross-links between different polymeric molecules in the dECM or implant. Crosslinking may be performed on the dECM ink or on the formed implant. Typically, the scaffold is cross-linked by one or more of the means selected from the group comprising: dehydrothermal (DHT) cross-linking; and chemical cross-linking. The crosslinking agent is typically a chemical crosslinking agent. Suitable chemical crosslinking agents and methods will be well known to those skilled in the art and include a glyoxal, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) or Glutaraldehyde.

In this specification, the term “GAG” should be understood to mean glycosaminoglycan, particularly sulphated glycosaminoglycans.

In this specification, the term “reduced GAG content” as applied to ECM from a given source should be understood to mean a GAG content that is reduced compared to natural ECM from the same source, for example less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5% GAG content of natural ECM. Methods of reducing GAG content include the use of buffers, detergents (such as Sodium dodecyl sulfate or Triton - X or Sodium deoxycholate) or other chemicals (e.g. chondroitinase ABC) known to reduce the sGAG content of tissues.

Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

Example 1 - Decell u la rised and solubilised meniscus tissue (Protocol 1)

Meniscus was harvested from 3 month old female pigs and diced into 1-2 mm pieces using a scalpel, followed by freeze-drying for 24 h at -10°C and cryo-milled in liquid nitrogen. The powder of meniscus tissue was immersed in 10 mM Tris-HCI buffer and went through 3 freeze-thaw (-80 to 37 °C) cycles with the buffer changed every cycle. The tissue was then treated with 1% triton-x100 solution for 24 h under rotation at room temperature. After washing with ultra-pure water, the tissue was treated with 10 mM Tris-HCI containing 0.15 M NaCI, 2 mM MgCh and 50 ll/rnl DNase (Sigma-Aldrich, Ireland) for 24 h at room temperature with gentle agitation, followed by 2 times of freeze-thaw cycles in 50 mM Tris- HCI buffer. The decellularized tissue was subsequently solubilized using a solution of 1500 ll/rnl of pepsin (Sigma-Aldrich, Ireland) in 0.5 M acetic acid under rotation for 24 h at room temperature. The tissue sample was then centrifuged at 2500g for 1 h to remove the insoluble material and the supernatant containing the solubilized tissue was transferred into a dialysis membrane (MWCO 12 - 14 kDa). The tissue sample was dialyzed against deionized water (5L) for 48 h maximum with 3 water changes before being freeze-dried. The obtained dry decellularized tissue samples were kept at -85 °C for long-term usage.

Example 2 - Decell ula rised and solubilised meniscus tissue (Protocol 2)

Meniscus was harvested, freeze dried and cryo-milled as specified in the Protocol 1. The freeze-dried tissue powder was then pre-treated with 0.2 M NaOH solution for 24 h at 4 °C with gentle agitation to extract the majority of sulphated glycosaminoglycan (sGAG). After that, the tissue was subsequently solubilized with pepsin as described in the Protocol 1 , followed by a centrifugation step to remove insoluble material. The supernatant was combined with a 5M NaCI solution to a final concentration of 0.8 M NaCI to preferentially salt precipitate collagen from the sample. The precipitated material was then solubilized again in 0.5 M acetic acid and the salt precipitation procedure was repeated a second time. The acid solubilized sample was then dialyzed against 0.02 M Na₂HPO₄for 24 h before being lyophilized. The lyophilized dry sponge was kept at -85 °C for long-term usage.

Example 3 - dECM Slurry/ink The lyophilized dECM was stored at -85 °C. To create the slurry/ink, the required amount of material (e.g. 70mg/mL) was weighed and dissolved into 0.5M of acetic acid containing the given NaCL concentration (e.g. 150mM). The solution was rotated at 4 °C for 48 hours prior use, to ensure complete dissolution. Before use, the pH of the slurry/ink was changed accordingly by dropwise adding a solution of NaOH 10M, while maintaining the 4 °C temperature of the sample. To ensure proper homogenization of the slurry/ink, some grains of phenol red were added to the solution.

Example 4 - Printed Meniscus Implant

The dECM dissolved ink at the proper pH and salt concentration as mentioned in the Example 3, was introduced into a printer cartridge, and centrifuged at 600 rpm for 5 min. Then, the cartridge was coupled to the thermostat printhead (15 °C) of the printer (REGENHU, Switzerland) before printing. For printing, a blunt 23G needle was used, and a speed of 35mm/s and an extrusion pressure of 0.18 - 0.30 MPa were selected.

The meniscus implant was designed with the commercial software from the same company (BioCad), layer by layer. The selected shape and size are similar to those found in adult native human medial meniscus: A C-shape of 46 mm large, 24mm wide and 9.5mm height. The printing pattern followed a circumferential configuration.

After printing, the implant was incubated for 4h at 37 °C before performing the crosslinking by introducing it into a solution of 0.5% glutaraldehyde in deionized water containing the same salt concentration as the ink and adjusted to pH11. After 24h of incubation at RT, the implant was rinsed with PBS and washed thoughtfully for 4 days.

Example 5 - Cast Meniscus Implant

A 3d printed clear resin meniscus shape (Form 3, Formlabs) was designed in Solidworks with the same size and shape as specified in Example 4. From this positive geometry a negative PDMS mould was formed to provide a suitable anatomically shaped mould for casting of the d-ECM.

The d-ECM slurry dissolved at the appropriate salt concentration (150 mM)and pH (11) as specified in Example 3, was introduced into the negative PDMS mould. Then, the slurry underwent a degassing process by introducing it into a vacuum chamber for 15 minutes, with a subsequent incubation for 4h at 37 °C. Once the embodiment was demoulded, it was crosslinked into a solution of 0.5% GA in DI water with the same salt concentration that the implant has (150mM), with an adjusted pH11. After 24h of incubation at RT, the implant was rinsed with PBS and washed thoughtfully for 4 days.

Generally, the invention uses casting or 3D printing of high concentration, decellularised meniscus ECM to produce implants that more closely mimic the structure, composition and mechanical properties of native meniscus tissue compared to existing implants. This is achieved by:

1. Improving the mechanical properties of 3D printed (or cast) constructs by adjusting the dECM concentration, pH and optionally NaCI concentration of the hydrogels I inks. To- date constructs printed using dECM inks have been soft and fragile. In addition, ECM or collagen-based scaffolds have been widely reported, but a common limitation of these scaffolds is their poor mechanical properties (e.g. the commercial Collagen Meniscus Implant (CM I®)), making them unsuitable for immediate load bearing applications. Herein, the main focus of this invention is to provide a novel strategy to print (or cast) mechanically functional implants for meniscus (or articular cartilage) tissue regeneration. These implants can be used as an alternative to meniscal allografts (or osteochondral allografts) for joint repair.

2. Improving the 3D printability of dECM inks by using a high concentration of ECM at specific pH levels. Previously, dECM inks were normally prepared at low concentration and neutral pH, e.g. 2% and pH 7.4, in order to maintain the cell viability when bioprinting with cells. However, the shape fidelity after printing was poor. In this invention, we use high concentration (for example 5-10%) dECM ink at high pH (e.g.11) to improve printability enabling the high resolution printing of large size acellular implants, as demonstrated in Figure 3, by using any of the two protocols developed for ECM decellularisation and solubilisation (P1 and P2 of Examples 1 and 2 above). Anatomically shaped meniscal implants can also be produced by casting the same dECM inks (at pH 11) into preformed moulds, as demonstrated in Figure 4.

3. Existing meniscus implants fail to recapitulate the native collagenous fibril distribution of the native meniscus, which determines its mechanical anisotropy. By selecting the appropriate pH, nozzle diameter and printing speed during the 3D printing of the dECM inks, the extent of collagen fiber alignment in the resulting print can be controlled (Figure 5 & Figure 6). Moreover, changing the quantity of NaCI added to the dECM ink also changes the conformation of the fibers on a micro-scale. (Figure 2).

4. 3D printing of ECM constructs with anisotropic collagen microstructure. The importance of scaffold/implant architecture in directing successful tissue engineering has been widely reported, however 3D printing of anisotropic ECM constructs remains challenging. In this invention, control over the fiber formation and alignment through 3D printing can be used to build scaffolds that recapitulate the native meniscus fiber arrangement. In fact, it has been demonstrated that tensile properties of printed dog bones increase due to the orientation of the deposited fibers (Figure 7). Therefore, by adjusting fiber alignment, a dECM implant can be fabricated with different zones containing different structural and mechanical properties, to provide an environment that would mimic the native one (Figure 9).

5. It is also possible to dry the 3D printed (or casted) dECM implants by performing snap freezing in liquid nitrogen (LN) after freeze drying (FD) or performing the freeze drying of the scaffolds directly. Thus, the fabricated implants can be more easily stored to improve their longevity and facilitate their handling and transportation. Furthermore, the altered architecture introduced by the drying process changes the elasticity of the implants, increasing it in the case of the casted ones, that are less likely to mechanically fail when loaded (Figures 10 and 11).

6. Identification of solubilisation and decellularisation methods for native meniscus tissue that result in the development of ECM hydrogel inks that enable high resolution 3D printing (Examples 1 and 2). Different decellularisation methods for tissues such as articular cartilage and meniscus have been reported in previous inventions and in the literature, however the printability of a hydrogel or ink prepared from such ECM products is either poor (e.g. lack of fidelity or resolution) or not reported. The problem is commonly addressed by mixing the dECM ink with a secondary hydrogel, producing a less biomimetic environment. As an alternative to 3D printing, the same dECM inks can also be cast into preformed moulds to produce ECM implants of specific shapes (e.g. the shape of the meniscus).

Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:

1. A cartilage or meniscus implant formed from decellularized cartilage extracellular matrix (dECM) ink, in which the dECM ink has a dECM concentration of 5 to 10% (w/v), wherein the dECM in the implant is chemically crosslinked, and wherein the dECM ink has a pH of 9 to 12.

2. A cartilage or meniscus implant according to Claim 1, in which the implant is freeze-dried and chemically crosslinking.

3. A cartilage or meniscus implant according to Claim 1 or 2, in which the dECM ink does not contain a second hydrogel.

4. A cartilage or meniscus implant according to any preceding Claim, in which the implant is fibrous and is formed by 3-D printing fibres with the dECM ink to form a fibrous 3-D construct.

5. A cartilage or meniscus implant according to any of Claims 1 to 3, in which the implant is formed by casting the dECM ink in a mould.

6. A cartilage or meniscus implant according to any preceding Claim, in which the dECM comprises 50 to 350 mM salt.

7. A cartilage or meniscus implant according to any preceding Claim, in which the implant is formed in the shape of a natural cartilage tissue.

8. A cartilage or meniscus implant according to any preceding Claim, in which the implant is formed in the shape of a meniscus tissue of the knee joint.

9. A cartilage or meniscus implant according to any preceding Claim, in which the implant is formed by 3-D printing, and in which the implant comprises a first 3-D printed fibre and a second 3-D printed fibre, in which the first 3-D printed fibre is compositionally and/or dimensionally different to the second 3-D printed fibre.

10. A cartilage or meniscus implant according to Claim 6, in which the first 3-D printed fibre comprises a first dECM ink and the second 3-D printed fibre comprises a second dECM ink, wherein the first and second dECM ink’s differ in at least one of the following: pH; dECM concentration, and salt concentration.

11. A cartilage or meniscus implant according to Claim 9 or 10, in which the first 3-D printed fibre has a different thickness to the second 3-D printed fibre.

12. A cartilage or meniscus implant according to any preceding Claim, in which the implant is formed by 3-D printing, and in which the implant comprises at least a first 3-D printed fibre and a second 3-D printed fibre, in which the first 3-D printed fibre is arranged in a first configuration and the second 3-D printed fibre is arranged in a second configuration that is different to the first configuration.

13. A cartilage or meniscus implant according to Claim 12, in which the implant is fibrous, and in which at least some of the printed fibres of the implant extend in a circumferential configuration around the implant.

14. A cartilage or meniscus implant according to any preceding Claim, for use in a method of repairing a high load bearing joint in a mammalian subject, in which the implant is surgically implanted into the high load bearing joint to replace some or all of the natural cartilage of the joint.

15. A cartilage implant according to any of Claims 1 to 13, for use in a method of Claim 14, in which the high load bearing joint is the knee, and in which the implant is a meniscus implant.

16. A method of making a decellularized extracellular matrix (dECM) ink comprising the step of mixing dECM with a solvent to provide an ink having a dECM concentration of 5% to 10% (w/v), wherein the dECM ink does not include a second hydrogel.

17. A method according to Claim 16, comprising a step of adjusting the pH of the ink to a pH of 9 to 12.

18. A method according to Claim 16 or 17, comprising a step of adding salt to the dECM and solvent to provide an ink having a salt concentration of 10 mM to 500 mM.

19. A method according to any of Claims 16 to 18, in which the dECM is formed by a process comprising the steps of: size-reducing ECM tissue (e.g. meniscal or cartilage tissue); pretreatment of the size-reduced tissue; digestion with pepsin; dialysis with a buffer; and freeze-drying.

20. A method of making a cartilage or meniscus implant comprising providing a decellularized extracellular matrix (dECM) ink, and forming an implant from the dECM ink comprising (a) casting the dECM ink in a mould or (b) 3-D printing one or more fibres from the dECM ink, and forming the implant from the one or more 3-D printed fibres.

21. A method according to Claim 20, in which the dECM ink does not comprise a second hydrogel.

22. A method according to Claim 20 or 21 , comprising freeze-drying the implant and then cross-linking of the freeze-dried implant.

23. A method according to any of Claims 20 to 22, in which the implant is formed by 3-D printing, in which the 3-D printing step comprises printing a first part of the implant using a first dECM ink and printing a second part of the implant using a second dECM ink, wherein the first and second dECM inks differ in a parameter selected from dECM concentration, pH, and salt concentration.

24. A decellularized extracellular matrix (dECM) ink comprising 5 to 10% dECM and solvent, wherein the dECM ink does not include a second hydrogel, and wherein the dECM ink has a pH of 9 to 12.

25. A decellularized extracellular matrix (dECM) ink comprising:

5 to 10% dECM (w/v);

10 mM to 500 mM salt; and solvent, wherein the dECM ink does not include a second hydrogel, and wherein the dECM ink has a pH of 9 to 12.