Surgical scaffold

Abstract

A surgical scaffold (100,200) made from a composition comprising a biocompatible material and a graphene-based material, wherein the graphene-based material comprises or consists of graphene, reduced graphene oxide (rGO) and / or functionalised graphene materials.

Description

Surgical ScaffoldTECHNICAL FIELDEmbodiments of the invention relate to a surgical scaffold, and in particular, though not exclusively, to a scaffold configured to mimic layered tissue structures found in joints.BACKGROUNDThe human body contains several types of joints, which provide articulated connections between bones in the body, and are configured to allow various degrees of movement. The knee is a joint which provides, in particular, an articulated connection between the femur and the tibia and a connection between the femur and the patella. In the knee joint, the femur and the tibia have complementary shapes which allows the femur and the tibia to move in relation to each other with a partially constrained path. The bone surfaces at the joint are protected by cartilage which provides a lubricated contact surface and ensures that the joint surfaces can slide easily over each other.The knee is particularly prone to injury and also to damage as a result of a number of conditions, such as osteoarthritis. When the knee is damaged to the extent that the function of the knee is compromised and / or that the pain suffered by a subject is too great, a surgical procedure known as knee replacement is often considered. Knee replacement can involve partial knee replacement (known as unicompartmental knee arthroplasty - UKA or patello-femoral joint replacement - PFJR) or total knee replacement (known as total knee arthroplasty - TKA). UKA involves replacing the femoral, tibial and I or patellar surfaces only in the compartment of the knee which is damaged (medial femorotibial or lateral femorotibial compartments or patellofemoral compartment), whereas TKA involves replacing the whole knee joint on both the femoral and the tibial side of the knee as well as the patellofemoral joint in some cases.Conventional knee replacement implants consist of three parts: a femoral component usually made of metal or rarely of ceramic; a tibial component made of metal (so-called metal-back) or a synthetic polymer material (typically polyethylene (PE)) or rarely of ceramic; and an insert made of a synthetic polymer material (typically polyethylene (PE)), configured to provide a contact surface between the femoral component and the tibial component. The insert is generally attached to the tibial side (metal-back or ceramic base), and provides a contact surface to cooperate with and act against the femoral component. in some cases, the tibial component and insert are made as one piece of polyethylene (known as an “all poly” tibia) thus generating a two-component implant that mimics a three-component implant.These parts are typically available in a range of pre-determined sizes.Prior to carrying out a knee replacement operation the surgeon will typically use some type of medical imaging (X-ray, CT, MRI, etc) and software to assess the patient’s knee and select the size or sizes of each component that are considered to fit best the bone anatomy of the patient.During the operation the surgeon will make cuts to parts of the femur and tibia and sometimes the patella in order to fit these components of pre-selected sizes and to correct any malalignment of the lower limb or in the joint itself.55723204-1Unfortunately, these metal and synthetic polymer implants wear out and, unlike the human body, do not have the ability to regenerate or repair themselves.Tissue engineering aims to produce materials, often including human cells, that can be implanted into humans to replace worn out or defective organs, such a hearts or livers. Ideally, these materials allow the growth of tissues that are just as natural tissues and so, if there is no disease state, can regenerate and repair themselves for the life of the individual just as our normal tissues do. Currently, combining biomaterial scaffolds with living stem cells for tissue regeneration is a main approach for tissue engineering [Luo et al. 2015], 3D “functional” scaffolds improve the regenerative potential of stem cells and are constructed to optimise biochemical, biophysical, and mechanical characteristics [Shadjou et al. 2017], The scaffold mimics the extra cellular matrix (ECM) that surrounds cells in the body so that cells can grow under the conditions that they normally encounter [Liao et al. 2015], The scaffold can also contain / deliver bioactive molecules to the cells / tissues to accelerate healing (growth) [Shadjou et al.2017], Tissue engineering has looked towards stem cells to produce new tissue as these cells can become a variety of mature cells and so can, with the correct environment, produce a complex tissue containing different cell types as required. Initial stem cell work used embryonic stem cells but these raise ethical as well as supply issues. Therefore, there has been a move to adult stem cells and particularly mesenchymal stem cells (MSCs) which are also known as multipotent cells. These exist in adult tissues of different sources, are self-renewable, multipotent, easily accessible and culturally expandable in- vitro with exceptional genomic stability and few ethical issues [Horwitz et al. 2005], These have a strong affinity to differentiate into the mesodermal cells (adipocytes, osteocytes and chondrocytes) [Ullah et al. 2015], They can be isolated from adipose (fat) tissue which makes them easy to harvest from a particular individual. The biggest stumbling block in the use of MSCs in tissue engineering is stem cell fate [Kenry et al. 2018], Conventionally, chemical inducers and growth factors are used as biochemical signals. However, stem cell behaviour heavily dependent on cell - substrate interactions with the ECM central to controlling cell behaviours [Kenry et al. 2018, Engler et al. 2006], Therefore, the material properties surrounding the stem cells: surface topography; stiffness; density; porosity are important [Kenry et al. 2018],Current treatments for cartilage damage are either reparative or restorative and include Mosaicplasty (Autologous Osteochondral plug transfer), Microfracture, Autologous Chondrocyte Implantation (ACI), Matrix-Induced Autologous Chondrodyte Implantation (MACI) or osteochondral autograft transfer. Mosaicplasty requires harvesting osteochondral plugs from healthy areas and transferring them into damaged areas of the knee. Mosaicplasty facilitates cartilage matrix but there is a lack of peripheral integration. Microfracture or subchondral drilling requires debridement of damaged cartilage in order to stabilise a rim and uses a drill or an awl to perforate the subchondral bone and produce a blood clot - subsequently fibrocartilage is generated which will cover the damaged area(s) over time. In Autologous Chondrocytes Implantation (ACI) Chondrocytes are first harvested, manipulated, expanded in culture and then reimplanted under a sutured periosteal flap. Autologous allograft transplantations are transplanted into cartilage defects after being shaped to it. Autologous chondrocyte implantation (ACI) had been advocated to improve inconsistent outcomes of microfractures [Goyal et al. 2013], ACI had obvious advantages to reduce immune and55723204-1infection complications and avoid large chondrocyte harvesting performed in mosaicplasty which is another traditional technique to treat cartilage damages [Bentley et al. 2012], However, some drawbacks of ACI have been identified such as the need for two surgeries, delayed weight bearing for at least 6 weeks, long recovery time up to 12 months, the need for further surgery such as osteotomies and the risk of hypertrophy of the cartilage flap [Peterson et al. 2000, Makris et al. 2015], This last adverse event is the most frequently reported after ACI explaining why alternative approaches utilizing artificial matrices have been tried. These artificial matrices are porcine membranes consisting of mixture of collagen type I and III or hyaluronic acid scaffold. However, these material increases the likelihood of an immune reaction to allogenic scaffold annihilating ACI benefits [Saris et al. 2008, Saris et al. 2009], Their use was considered off-label in the USA [Makris et al. 2015] but was FDA approved in December 2016 [US FDA 2016], The complexity of ACI and contraindications in wide clinical application have driven the development of matrix-assisted chondrocyte implantation (MACI), which uses scaffolds to provide mechanical stability and support chondrogenesis. MACI uses scaffolds plus either primary articular chondrocytes or bone-marrow-derived cells (MSCs). MACI also requires two surgical procedures as well but has lower rate of hypertrophy of the graft compared to ACI. Although case series of MACI have had promising clinical outcomes, in terms of reproducibility, safety, surgical simplicity, reduced invasiveness, intraoperative time and histological results, the superiority of MACI over existing techniques remains unproven [Marlovits et al. 2012; Ebert et al. 2020] despite encouraging outcomes [Ebert et al. 2017, Wondrasch et al. 2009], Large prospective randomized trials with long follow-up periods are still necessary to confirm preliminary benefits. More recent techniques such as AMIC (Autologous matrix-induced chondrogenesis) combining microfracture and glued or sutured matrix with collagen I and III into the defect has been popular because of the simplicity and cost-effectiveness [Benthien & Behrens 2011 , Tradati et al. 2020], However, AMIC merits further studies to identify the quality of neocartilage formation, the reproducibility of the data and any potential pitfalls associated with the technique.Current practice recommends: microfracture and chondroplasty for lesion <1.5cm2; microfracture, mosaicplasty, allograft, ACI for lesions < 4 cm2and possibly up to 8 cm2; whereas only allograft and possibly ACI are recommended for lesion >8 cm2. Outcomes are variable and none of these techniques have shown significant improvements compared to others.However, all these techniques showed better anatomical and functional outcomes when associated to limb alignment correction such as osteotomies. There is also direct cartilage transplant as with the BioUni OATS (OsteoArticular Transfer System) Instrument Set (Arthrex, USA) where surgeons can replace damaged cartilage with a single, elliptical piece of viable, hyaline cartilage.For newer treatments there has been a focus on putting stem cells, particularly MSCs derived either from bone marrow or adipose tissues, directly into defects in knee joints either as a paste, a soaked sponge or in scaffolds. The current state of the art in available products to repair cartilage damages includes those made of cells and acellular biomaterials. A further development of ACI and MACI are scaffold-based approaches which have potential major advantages to scaffold-free techniques in increasing control to better fill the cartilage defect; fewer donor site complications; a less technically55723204-1challenging procedure; and shorter postoperative recovery time due to increased graft stability. Additionally, because the chondrocytes are cultured in a 3D environment, they are less prone to dedifferentiation in fibroblasts and therefore produce a more hyaline- like cartilage [Caron et al. 2012], Furthermore, in-vitro culture prior to implantation might help to maintain quality control of scaffold-based repair. Hyaluronic-acid-based scaffolds is intended to promote and maintain the chondrocyte phenotype and collagen type II synthesis during in vitro culture, as well as after implantation [Bian et al. 2011], Scaffold based ACI / MACI for cartilage regeneration is approved in Europe and the FDA approved MACI in December 2016. Furthermore, athletes treated by microfracture required at least 8 months of recovery before returning to the field, whereas those receiving hyaluronic-acid-based MACI required 12.5 months [Kon et al. 2011], Within the cell based treatments are: BioCartilage (Arthrex, USA) which is a desiccated cartilage particulated allograft mixed with a biologic like bone marrow concentrate (BMC) or platelet rich plasma (PRP) that is pressed into the defect and sealed with fibrin glue; DeNovo NT (Zimmer Biomet, USA) is a human tissue allograft of juvenile hyaline cartilage pieces with viable chondrocytes; Cartiform (Arthrex, USA) which is a cryopreserved osteochondral allograft with chondrocytes, chondrogenic growth factors and Extra Cellular Matrix (ECM) proteins; NeoCart (Histogenics, Massachusetts, USA) implants are an autologous cartilage tissue; CellcoTec (CellCoTec BV Bilthoven, The Netherlands) which is a bilayer bacterial nanocellulose bio-degradable, load bearing, mechanically functional scaffold which is seeded with patients own cartilage and bone marrow; ProChondrix CR (Allosource, Stryker, USA) is a laser-etched, fresh cryopreserved osteochondral allograft which contains growth factors to maintain healthy cartilage and facilitate chondrocyte functionality. These all focus on the repair of focal cartilage defects and aim to support cell viability, adherence and migration. BioCartilage® has been used in an equine model and DeNovov® in human talar (ankle joint) cartilage lesions. NeoCart® has been shown to be safe and effective in the human knee when compared to microfracture with repair tissue being durable and evolving over. However, these techniques can only repair small size focal lesions (as given above) and would require further assessment to define its effectiveness into the range of possible treatment. Despite the rapidly growing market in the field of biologic therapies and products to repair osteocartilaginous damages, most of the developed techniques can only repair small size focal lesions and still require further clinical long-term assessment to prove their efficacy in peer-reviewed literature. Only a few of these products are actually fully FDA approved such as MACI and Collagen Meniscal Implant for instance whereas others are under review from the Center for biologies evaluation and research (CBER) or the centre for devices and radiological health (CDHR) of the FDA.There also exist acellular scaffold-only based treatments such as: Agili-C® (CartiHeal, USA) which is a cell free resorbable bi-phasic scaffold made of natural inorganic calcium carbonate; Cartipatch (TBF Tissue Engineering, France) which is solid scaffold with an agarose-alginate matrix. These aim to encourage cells (stem cells, chondrocytes) to populate the scaffold so that cartilage and subchondral bone are regenerated. These are also aimed at small focal lesions. Agili-C™ has been used in larger lesions in Osteoarthritis (OA) but these results are not published. Cartipatch® was used in much larger lesions (2.5 - 7.5cm2) and was shown not to be as good a mosaicplasty [Clave et al. 2016], Allografts or acellular scaffolds both have the drawback55723204-1that they require cells to repopulate the implants which is not always complete, particularly when there are large volumes. This gives uncertain long-term viability.There is still no panacea to treat cartilage damage and underneath pathologic structures (subchondral bone and bony architecture). The multiplicity of techniques to treat bony cartilage defects, including new approaches such as scaffold-free technology based on generating spheroids of autologous chondrocytes “chondrospheres” demonstrates limitations in any of them. The clinical efficacy of chondrospheres is currently being investigated in phase III controlled clinical trial in Europe [Meyer et al. 2012, ClinicalTrials.gov [Internet], Bethesda (MD): National Library of Medicine (US); 2014. co. don AG Efficacy and Safety Study of co. Don Chondrosphere to Treat Cartilage Defects U.S. National Institutes of Health. Available from: http: / / clinicaltrials.gov / show / NCT01222559 ] [Hoburg A, Niemeyer P, Laute V, Zinser W, Becher C, Kolombe T, Fay J, Pietsch S, Kuzma T, Widuchowski W, Fickert S. Matrix- associated autologous chondrocyte implantation with spheroid technology is superior to arthroscopic microfracture at 36 months regarding activities of daily living and sporting activities after treatment. Cartilage. 2021 Dec;13(1_suppl):437S-48S.]Further work has been done to produce layered scaffolds that will encourage the infiltration, proliferation and growth of the correct cell type at the correct point in the construct. Levingstone et al.

[2014] created a layered construct using a freezing drying iterative layering technique which mimicked the structure of healthy osteochondral tissue: a “bone” layer ECM of type I collagen and hydroxyapatite (HA), an intermediate layer ECM of type I collagen, type II collagen and HA and a “cartilage” layer ECM of type I collagen, type II collagen and hyaluronic acid. These different layers within the scaffold were designed to give the correct environment for differentiation of cells towards the correct type for each region of the tissue. The implant structure had high levels of porosity (>97%) and in in-vitro culture cells migrated throughout the scaffold [Levingstone et al. 2014],3D bioprinting has been used to produce bone [Turnbull et al. 2018a] and cartilage [Kang et al. 2016, Turnbull et al. 2018b, Zhou et al. 2018], Human chondrocytes can be harvested and bioprinted in hydrogels (such as alginate, gelatin, and nanocellulose and other ECM) while cells maintain their high viability and geneexpression profiles [Schuuram et al. 2013, Kang et al. 2016, Coates et al. 2012], Similarly, amniotic fluid-derived stem cells have been isolated and 3D bioprinted into bones [Pishnamazi et al. 2024], However, to produce a knee implant requires bone, subchondral bone and organised hyaline cartilage. Knee implants also need to be strong enough to allow weight-bearing immediately post-surgery. This is due to the more recent advances in enhanced recovery after surgery which has shown that early mobilisation i.e. up and walking within 24 hours of surgery, is important for rapid and full recovery [McDonald et al. 2012], Usually 3D bioprinted structures containing only cells do not have strength to start with and need to be matured for cells to produce ECM and a stronger tissue. To strengthen 3D bioprinted structures scaffolds are widely used [prior art, Kang et al. 2016], The problem with these is often their biocompatibility, degradation rates, strength and ability to maintain and support cells growth and stem cell differentiation. The co-printing or simultaneous printing of scaffolds (PCL / borate glass) and cells (adipose MSCs) with a two-nozzle printer has been shown to provide an environment where the cells proliferate and osteogenic differentiation takes place on the scaffold55723204-1[Murphy et al. 2017], The use of graphene in scaffolds seeded with cells have been shown to improve cell proliferation and attachment [Wang, W et al. 2016],Graphene has been shown to be an excellent material for cell maturation. Different types of graphene cause stems cells to differentiate. To produce bone need stems cells to differentiate into osteoblasts. Tang et al. showed increased surface roughness of graphene oxide (GO) enhanced bone matrix formation with MSCs [Tang et al. 2012], Patterned graphene-based films enhanced osteogenic differentiation [Kim et al. 2015], 3D graphene-based scaffolds for osteogenic differentiation showed pore size and porosity were important [Crowder et al. 2013], GO mixed with hydrogel (PEGDA) showed increased osteogenesis [Noh et al. 2017], Vertical carbon nano walls on substrate promoted osteogenic cell differentiation and mineralisation [Borghi et al. 2018], GO improves cells (pre-osteoblasts) adhesion, differentiation, proliferation and calcium phosphate deposition [Depan et al. 2011], For hASCs (adipose derived stem cells) GO-coated substrate improved adhesion and osteogenesis but suppressed chondrogenesis [Kim et al. 2013], Graphene foam (GF) support hMSCs and induce spontaneous osteogenic differentiation [Verre 2018] but this used nickel so has some problems with biocompatibility. 3D rGO aerogels functionalised by chitosan and mineralised by incubation with simulated body fluids to mimic formation of natural bone. This promoted osteogenic differentiation [Asha et al. 2018], 0.5% by weight GO and gelatin hydroxyapatite scaffolds induced osteogenic differentiation of human adipose derived mesenchymal stem cells without providing standard supplements (dexamethasone, L-ascorbic acid and p glycerophosphate) and the level of osteogenic differentiation of stem cells was comparable to those cultured on Gelatin-Hydroxyapatite (GHA) scaffolds with these osteogenic supplements [Nair et al. 2015], A bioinspired surface of GO-gelatin (GO-Gel) showed higher cellular activities (MC3T3-E1 cells) such as cell adhesion, cell proliferation, and alkaline phosphatase activity (ALP) compared with GO or glass surface and so promoted the osteogenic differentiation of MC3T3-E1 cells [Liu et al. 2014], Evidence of mineralization corroborated the idea that a native osteoid matrix was ultimately deposited [Liu et al. 2014], A bioglass graphene composite (58S bioglass and 0.5%by weight graphene) scaffold showed the scaffold surface was covered by a thick and well-formed hydroxyl carbonated apatite (HCA) layer after immersion in SBF for 7 days and MG-63 cells colonized and grew favourably over 7 days incubation [Gao et al. 2014], MC3T3-E1 (mouse osteoblastic) cells attached and grew on the surfaces of nanofibrous biocomposite scaffolds of poly(vinyl alcohol) (PVA) and GO prepared by using electrospinning method, and the adding of GO did not affect the cells' viability [Qi et al. 2013], Luo et al.

[2015] showed that GO-doped poly(lactic-co- glycolic acid) (PLGA) nanofiber mats with three-dimensional porous structure and smooth surface accelerated the human MSCs adhesion and proliferation versus pure PLGA nanofiber and induced the osteogenic differentiation.To produce cartilage this needs stem cells to differentiate into chondrocytes with a focus on producing hyaline cartilage rather than fibrocartilage. When using MSCs and graphene flakes to form graphene cell biocomposites increasing the concentration of graphene and porous GO correlated positively with chondrogenic differentiation [Lee et al. 2015], Liao et al. [2015 published in Scientific Reports] implanted a hybrid polymer / GO scaffold that was cultured with cartilage cells into osteochondral defects in rabbits (rabbit knees). At 18 weeks post implant this showed good repair with the cartilage55723204-1embedded with the surrounding tissue and there being cartilage and subchondral bone in the defect. Yocham et al.

[2018] shown that chondrocyte progenitor cells were viable on graphene foam (GF) made by CVD with cells adhering to the GF within 24 h of cell culture, spanning the pores of the GF by day 7 filling the pores by day 14 with successful cell proliferation throughout the 3D bioscaffold at 28 days incubation.The use of graphene allows signalling to ensure the correct cells are generated at correct point and the strength of scaffold could allow almost immediate implantation of implant. The possibility of immediate implantation means that the implant and cells will “see” the correct mechanically signalling via joint loading for that individual so that correct tissues are formed as per Liao et al.

[2015] ,CN107789667A shows the use of graphene oxide with glucan and crystal cellulose to generate a scaffold that promotes the regeneration of bone or cartilage. IN05941CH2014A shows the use of graphene oxide with hydroxyapatite to produce scaffolds which support cell adhesion and proliferation, viability and proliferation and also mineralisation showing they are osteoconductive. US9433682B2 shows that graphene with hydrogels, chitosan and PEGDA (ADD) forms a scaffold that encourages both the osteogenic and chondrogenic differentiation of bone marrow MSCs. W018204702A1 shows that phosphate functionalized graphene oxide-based scaffolds encourage the production of bone from MSCs. US2017130194A has scaffolds coated with graphene oxide and then seeded with stem cells which have been shown to cause cells to differentiate in the presence of culture mediums which may or may not contain growth factors.W02020205831A1 discloses porous scaffolds which comprise microspheres made of a biodegradable polymer mixed with a graphene-based material. LIS2021180018A1 discloses a composition for promoting stem cell differentiation using a progenitor cell culture solution (e.g., human adipose mesenchymal stem cells) and a multilayer graphene film. The multilayer graphene film is a laminate of 2-10 layers of graphene, where the surface may be patterned using electron beam lithography, photolithography, or the like. CN110452397A discloses a 3D graphene foam polysaccharide-based hydrogel composite scaffold. CN111944750A relates to a 3D annular cell scaffold for use in culturing cells, such as mesenchymal stem cells, which comprises of a graphene 3D annular cell scaffold prepared from a copper / nickel template using a chemical vapour deposition method. CN112190755A discloses a scaffold material for bone tissue engineering, such as to promote formation of new bone tissues in vivo, which comprises a chitosan derivative, graphene oxide and Bianstone powder. - CN112704765A discloses a chitosan-graphene oxide composite gel and methods of preparation thereof. US2013230496A1 relates to a hydrogel composition comprising graphene, chitosan and polyethylene diacrylate (PEGDA). WO19198986A1 relates to a cell culture scaffold for promoting stem cell differentiation using embryonic or adult stem cells, which is formed from multilayer graphene film of 2 to 10 graphene layers. W018204702A1 relates to a method of functionalising graphene oxide and a device comprising a bone scaffold formed using phosphate functionalised graphene oxide and metal ions. US20190022279A1 relates to a tuneable scaffold for tissue regeneration including a first medium composed of bone particles with or without organic components, a second medium composed of a natural or synthetic biocompatible / biodegradable polymer, and a third medium comprising rapidly dissolving polymers in a solvent.55723204-1Graphene has been shown to be biocompatible [Nair et al. 2015, Xie et al. 2015, Kenry et al. 2018, Yocham et al. 2018, Tamayo Marin et al. 2019], It has also been shown that GO sheets can be degraded by neutrophils so graphene can be biodegradable [Mukherjee et al. 2018], Graphene flakes can be biodegraded by myeloperoxidase secreted by activated neutrophils or by recombinant myeloperoxidase therefore pristine graphene can be degraded by immune system - the body may be able to fully remove material after it has served its function [Kurapati et al. 2018], Degradation of polyvinyl alcohol (PVA) / chitosan (CS) / GO scaffolds in simulated biological fluid showed weight loss of 70% at 14 days [Tamayo Marin et al. 2019],Graphene can be used to enhance existing scaffolds to make their properties more suitable. Graphene has been used to enhance strength of hydrogels (basic scaffold for tissue engineering) without affecting cytotoxicity towards osteoblasts [Tadyszak et al. 2018], Liao et al. generated a biodegradable scaffold containing standard biodegradable scaffold polymers with GO with 150-200 pm pores which approached the loads for human walking (0.48MPa when range for human movement is 0.5 - 8 MPa). The scaffold degraded within two months when implanted in rats [Liao et al. 2015], A bioglass 58-S graphene composite (interconnected pores approximately 0.8 pm (800 nanometres) in size and isotropic in their distribution) showed the incorporation of 0.5% by weight graphene improved the compressive strength by 105% from 23.7 ± 3.9 MPa to 48.7 ± 3.2 MPa for and the fracture toughness by 38% from 1.41 ± 0.07 to 1.94 ± 0.10 MPa m1 / 2but higher levels of graphene did not add further improvements [Gao et al. 2014], The same was shown by Qi et al.

[2013] with the tensile strength and elasticity modulus of nanofibrous biocomposite scaffolds of poly(vinyl alcohol) (PVA) and graphene oxide (GO) prepared by using electrospinning method increased when the content of GO was lower than 1% by weight, but decrease when GO went up to 3 and 5% by weight [Qi et al. 2013], The inclusion of reduced graphene oxide (rGO) into polycaprolactone (PCL) at 0.5%wt into a 3D printed scaffold increased compressive strength by 185% (from 2.9MPa to 5.4MPa) and stiffness by 150% (from 28.8MPa to 42.8MPa) [Seyedsalehi et al. 2020], These scaffolds were shown in-vitro with human ADSCs to be cytocompatible, supporting cell growth and viability [Seyedsalehi et al. 2020],It is also possible to create drug eluting scaffolds, with antibiotics, growth factors or pain relief bound to the scaffold or hydrogel element allowing for controlled release over time. Huang et al.

[2014] reviewed different scaffold materials such as PCL, alginate and gelatin as platforms for elution of bioactive molecules such as TGF-p, VEGF and BMP-2 for use in bone and cartilage engineering. Rostami et al.

[2020] produced an electrospun PCL-GO-Dexamethasone composite which showed improved osteogenic differentiation of MSC. Foox and Zilberman

[2015] showed the propensity of gelatinbased biomaterials for drug encapsulation and elution. The literature suggests that there is a wide scope for incorporating drugs within scaffolds to further improve their biocompatibility.It is an object of the present invention to mitigate or alleviate one or more of the disadvantages associated with the prior art.55723204-1SUMMARYExamples according to the disclosure may be formed using an additive manufacturing process. A common example of additive manufacturing is 3D printing; however, other methods of additive manufacturing are available. Rapid prototyping or rapid manufacturing are also terms which may be used to describe additive manufacturing processes.As used herein, “additive manufacturing” (‘AM’) refers generally to manufacturing processes wherein successive layers of material(s) are provided on each other to “buildup” layer-by-layer or “additively fabricate”, a three-dimensional component. This is compared to some subtractive manufacturing methods (such as milling or drilling), wherein material is successively removed to fabricate the part. The successive layers generally fuse together to form a monolithic component which may have a variety of integral sub-components. In particular, the manufacturing process may allow an example of the disclosure to be integrally formed and include a variety of features not possible when using prior manufacturing methods.Additive manufacturing methods described herein enable manufacture to any suitable size and shape with various features which may not have been possible using prior manufacturing methods. Additive manufacturing can create complex geometries without the use of any sort of tools, molds or fixtures, and with little or no waste material. Instead of machining components from solid billets of plastic or metal, much of which is cut away and discarded, the only material used in additive manufacturing is what is required to shape the part.Suitable additive manufacturing techniques in accordance with the present disclosure include, for example, Fused Deposition Modeling (FDM), Selective Laser Sintering (SLS), 3D printing such as by inkjets and laserjets, Sterolithography (SLA), Direct Selective Laser Sintering (DSLS), Electron Beam Sintering (EBS), Electron Beam Melting (EBM), Laser Engineered Net Shaping (LENS), Electron Beam Additive Manufacturing (EBAM), Laser Net Shape Manufacturing (LNSM), Digital Light Processing (DLP), Continuous Digital Light Processing (CDLP), Direct Selective Laser Melting (DSLM), Selective Laser Melting (SLMMaterial Jetting (MJ), NanoParticle Jetting (NPJ), Drop On Demand (DOD), Binder Jetting (BJ), Multi Jet Fusion (MJF), Laminated Object Manufacturing (LOM) and other known processes.Additive manufacturing processes typically fabricate components based on three-dimensional (3D) information, for example a three-dimensional computer model (or design file), of the component.Accordingly, examples described herein not only include products or components as described herein, but also methods of manufacturing such products or components via additive manufacturing and computer software, firmware or hardware for controlling the manufacture of such products via additive manufacturing.The structure of one or more parts of the product may be represented digitally in the form of a design file. A design file, or computer aided design (CAD) file, is a configuration file that encodes one or more of the surface or volumetric configuration of the shape of the product. That is, a design file represents the geometrical arrangement or shape of the product.Design files can take any now known or later developed file format. For example, design files may be in the Stereolithography or “Standard Tessellation Language” (.stl)55723204-1format which was created for stereolithography CAD programs of 3D Systems, or the Additive Manufacturing File (.amf) format, which is an American Society of Mechanical Engineers (ASME) standard that is an extensible markup-language (XML) based format designed to allow any CAD software to describe the shape and composition of any three- dimensional object to be fabricated on any additive manufacturing printer.Further examples of design file formats include AutoCAD (.dwg) files, Blender (.blend) files, Parasolid (,x_t) files, 3D Manufacturing Format (,3mf) files, Autodesk (3ds) files, Collada (.dae) files and Wavefront (.obj) files, although many other file formats exist.Design files can be produced using modelling (e.g. CAD modelling) software and / or through scanning the surface of a product to measure the surface configuration of the product.Once obtained, a design file may be converted into a set of computer executable instructions that, once executed by a processer, cause the processor to control an additive manufacturing apparatus to produce a product according to the geometrical arrangement specified in the design file. The conversion may convert the design file into slices or layers that are to be formed sequentially by the additive manufacturing apparatus. The instructions (otherwise known as geometric code or “G-code”) may be calibrated to the specific additive manufacturing apparatus and may specify the precise location and amount of material that is to be formed at each stage in the manufacturing process. As discussed above, the formation may be through deposition, through sintering, or through any other form of additive manufacturing method.The code or instructions may be translated between different formats, converted into a set of data signals and transmitted, received as a set of data signals and converted to code, stored, etc., as necessary. The instructions may be an input to the additive manufacturing system and may come from a part designer, an intellectual property (IP) provider, a design company, the operator or owner of the additive manufacturing system, or from other sources. An additive manufacturing system may execute the instructions to fabricate the product using any of the technologies or methods disclosed herein.Design files or computer executable instructions may be stored in a (transitory or non-transitory) computer readable storage medium (e.g., memory, storage system, etc.) storing code, or computer readable instructions, representative of the product to be produced. As noted, the code or computer readable instructions defining the product that can be used to physically generate the object, upon execution of the code or instructions by an additive manufacturing system. For example, the instructions may include a precisely defined 3D model of the product and can be generated from any of a large variety of well-known computer aided design (CAD) software systems such as AutoCAD®, TurboCAD®, DesignCAD 3D Max, etc. Alternatively, a model or prototype of the component may be scanned to determine the three-dimensional information of the component.Accordingly, by controlling an additive manufacturing apparatus according to the computer executable instructions, the additive manufacturing apparatus can be instructed to print out one or more parts of the product.In light of the above, embodiments include methods of manufacture via additive manufacturing. This includes the steps of obtaining a design file representing the product and instructing an additive manufacturing apparatus to manufacture the product in assembled or unassembled form according to the design file. The additive55723204-1manufacturing apparatus may include a processor that is configured to automatically convert the design file into computer executable instructions for controlling the manufacture of the product. In these embodiments, the design file itself can automatically cause the production of the product once input into the additive manufacturing device. Accordingly, in this embodiment, the design file itself may be considered computer executable instructions that cause the additive manufacturing apparatus to manufacture the product. Alternatively, the design file may be converted into instructions by an external computing system, with the resulting computer executable instructions being provided to the additive manufacturing device.Given the above, the design and manufacture of implementations of the subject matter and the operations described in this specification can be realized using digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed in this specification and their structural equivalents, or in combinations of one or more of them. For instance, hardware may include processors, microprocessors, electronic circuitry, electronic components, integrated circuits, etc. Implementations of the subject matter described in this specification can be realized using one or more computer programs, i.e., one or more modules of computer program instructions, encoded on computer storage medium for execution by, or to control the operation of, data processing apparatus. Alternatively, or in addition, the program instructions can be encoded on an artificially generated propagated signal, e.g., a machine-generated electrical, optical, or electromagnetic signal that is generated to encode information for transmission to suitable receiver apparatus for execution by a data processing apparatus. A computer storage medium can be, or be included in, a computer-readable storage device, a computer-readable storage substrate, a random or serial access memory array or device, or a combination of one or more of them. Moreover, while a computer storage medium is not a propagated signal, a computer storage medium can be a source or destination of computer program instructions encoded in an artificially generated propagated signal. The computer storage medium can also be, or be included in, one or more separate physical components or media (e.g., multiple CDs, disks, or other storage devices).Although additive manufacturing technology is described herein as enabling fabrication of complex objects by building objects point-by-point, layer-by-layer, typically in a vertical direction, other methods of fabrication are possible and within the scope of the present subject matter. For example, although the discussion herein refers to the addition of material to form successive layers, one skilled in the art will appreciate that the methods and structures disclosed herein may be practiced with any additive manufacturing technique or other manufacturing technology.According to a first aspect, there is provided a surgical scaffold, wherein the surgical scaffold is made from a composition comprising a biocompatible material and a graphene-based material, and wherein the surgical scaffold has a first region having a first set of mechanical properties and / or structural properties, and a second region having a second set of mechanical properties and / or structural properties.The biocompatible material may comprise a polymeric material. The biocompatible material may comprise a biodegradable material. Typically, the biocompatible material may comprise a polymeric material, which may comprise one or55723204-1more polymers selected from the list consisting of polycaprolactone, polylactic acid, poly(lactic-co-glycolic) acid, polyglycolide, polylactide, polyhydroxobutyrate, chitosan, and hyaluronic acid.Typically, the polymeric material may comprise polycaprolactone (PCL). Advantageously, polycaprolactone is biologically inert and biodegradable and therefore well suited for the present application.The graphene-based material may comprise one or more graphene compounds or graphene derivatives selected from the list consisting of graphene, graphene oxide, reduced graphene oxide, and functionalised graphene materials.Typically, the graphene-based material may comprise reduced graphene oxide (rGO). Advantageously, rGO may help improve the biocompatibility of the scaffold and / or may increase mechanical strength.The surgical scaffold may comprise a plurality of regions having different mechanical and / or structural properties.The surgical scaffold has a first region having a first set of mechanical and / or structural properties, and a second region having a second set of mechanical and / or structural properties. The surgical scaffold may have a third region having a third set of mechanical and / or structural properties.Typically, the first and second sets of mechanical and / or structural properties may be different.Typically, the first, second and third sets of mechanical and / or structural properties may be different.Advantageously, the scaffold, e.g. the plurality of regions thereof, may be designed to mimic an anatomical part of the body, typically a bone structure.The scaffold, e.g. the plurality of regions thereof, may be designed to mimic two or more, e.g. three, natural layers of a joint structure, e.g. of a bone structure, typically bone, subchondral bone and cartilage.The scaffold, e.g. the plurality of regions thereof, may be designed to mimic a bone structure of the knee.Advantageously, the scaffold may comprise a plurality of regions or layers designed to mimic a bone structure of the knee. The first, second and third regions may typically define a first layer, a second layer and a third layer. The first, second and third layers may be designed to mimic a bone structure, typically bone, subchondral bone and cartilage.Advantageously, the plurality of regions, e.g. plurality of layers, may be integrated and / or may form or may define a unitary structure.The plurality of regions or layers, e.g. first region, second region, and third region, may form or may define a unitary or one-piece structure. By unitary or one-piece, it is meant that the individual regions or layers are not manufactured separately or subsequently joined together, but are manufactured as a single scaffold so as to define a unitary structure.Each region may be configured to promote growth of an associated type of tissue structure.The first region may be configured to promote growth of a cartilage-like structure.The second region may be configured to promote growth of a subchondral bonelike structure.55723204-1The third region may be configured to promote growth of a bone-like structure.The first set, second set and / or third set of mechanical and / or structural properties may include porosity, size of pores, concentration of graphene-based material (e.g. rGO), polymer strand thickness, and / or polymer strand direction.The properties of each region, e.g. of the first, second and / or third regions, may be selected to promote growth of associated type of cellular material so as to yield an associated bone structure, e.g. bone, subchondral bone or cartilage.The ideal properties for each cell type (human chondrocytes and osteoblasts) may be typically determined by systematic studies of cell growth and extracellular matrix deposition in scaffolds with varying porosity and rGO concentrations.The properties of each region may be achieved by adjusting one or more of the following parameters during manufacture, e.g. printing, of each region or layer: porosity, size of pores, concentration of graphene-based material (e.g. rGO), polymer strand thickness, and / or polymer strand direction.The third region, which may be configured to promote growth of bone-like structure, may have one or more of the following properties: a porosity in the range of about 60% to about 90%, e.g. about 70% to about 90%; a pore size, e.g. average pore size 400pm, in the range of about 350 to about 500pm; a concentration of graphene-based material, e.g. rGO 0.1 %, in the range of about 0.05% to 0.15% by weight, based on the total weight of the scaffold in the first region;- polymer strands having a diameter, e.g. average diameter 400pm, in the range of about 350-450pm.The second region, which may be configured to promote growth of subchondral bone-like structure, may have one or more of the following properties: a porosity of in the range of about 60% to about 90%; a pore size, e.g. average pore size 300pm, in the range of about 100 to 500 pm; optionally, in the range of about 250 to about 350pm; a concentration of graphene-based material, e.g. rGO, in the range of about 0.2% to about 0.3% by weight, based on the total weight of the scaffold in the second region;- polymer strands having a diameter, e.g. average diameter, in the range of about 250-300pm.The first region, which may be configured to promote growth of cartilage-like structure, may have one or more of the following properties: a porosity of in the range of about 60% to about 90%; a pore size, e.g. average pore size 200pm, in the range of about 100 to about 250 pm; a concentration of graphene-based material, e.g. rGO, in the range of about 0.4% to about 0.6% by weight, based on the total weight of the scaffold in the third region;- polymer strands having a diameter, e.g. average diameter, in the range of about 50-200pm.55723204-1In some examples, the scaffold detailed herein may have or comprise the same mechanical properties and / or structural properties throughout the scaffold. In some examples, the scaffold detailed herein may have or comprise uniform mechanical properties and / or structural properties. By way of example, the scaffold may comprise substantially uniform mechanical properties and / or structural properties throughout the scaffold.In one example, the scaffold detailed herein may comprise or consist of one or more properties selected from: a. an average pore size of about 100 to about 800 pm; optionally, in the range of about 200 to about 800 pm; b. a concentration of graphene-based material in the range of about 0.05% to about 1% by weight; c. polymer strands having a diameter in the range of about 100 to about 500pm.In one example, the properties of the scaffold detailed herein detailed herein may comprise or consist of: a. an average pore size of about 100 to about 800 pm; optionally, in the range of about 200 to about 800 pm; b. a concentration of graphene-based material in the range of about 0.05% to about 1% by weight; c. polymer strands having a diameter in the range of about 100 to about 500pm.In one example, the scaffold detailed herein may comprise or consist of one or more properties selected from: a. an average pore size of about 400 pm; b. a concentration of graphene-based material of about 0.1% by weight; c. polymer strands having a diameter in the range of about 400pm.In one example, the properties of the scaffold detailed herein may comprise or consist of: a. an average pore size of about 400 pm; b. a concentration of graphene-based material of about 0.1% by weight; c. polymer strands having a diameter in the range of about 400pm.The graphene-based material may comprise or consist of graphene, graphene oxide, reduced graphene oxide (rGO) and / or functionalised graphene materials. The graphene-based material may comprise or consist of graphene, reduced graphene oxide (rGO) and / or functionalised graphene materials. In some examples, the graphene-based material comprises or consists of reduced graphene oxide (rGO). In some examples, the graphene-based material comprises or consists of graphene, such as graphene (e.g. CAS-No: 7782-42-5), graphene (e.g. CAS-No: 7782-42-5) dispersed in water or a functionalised graphene material dispersed in water / water suspension.As described herein, the biocompatible material may comprise or consist of polycaprolactone (POL).55723204-1In one example, the graphene-based material may comprise or consists of reduced graphene oxide and the biocompatible material may comprise or consist of polycaprolactone (PCL).In one example, the graphene-based material comprises or consists of graphene, such as graphene dispersion in water (CAS-No: 7782-42-5), and the biocompatible material may comprise or consist of polycaprolactone (PCL).The composition of the scaffold detailed herein may further comprise a carbohydrate, such as a mono-, di-, oli- or polysaccharide. The scaffold may comprise a monosaccharide such as glucose, fructose, galactose or the like. Preferably, the carbohydrate may comprise or may consist of glucose. Advantageously, and without wishing to be bound by theory, the inclusion of a carbohydrate such as glucose may have a positive effect on cell viability, such as in use following implantation, and / or promote degradation of the scaffold.In one example, the composition may comprise the carbohydrate, e.g. glucose, at a concentration of at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% by weight. In one example, the composition may comprise glucose at a concentration of at least 30% by weight. In one example, the composition may comprise glucose at a concentration of about 30% by weight.The composition may comprise the carbohydrate, e.g. glucose, at a concentration of up to 50% by weight.In some examples, the composition may comprise the carbohydrate, e.g. glucose, at a concentration of between 10% to 50% by weight.The composition may comprise the carbohydrate, e.g. glucose, at a concentration of about 20-40 wt%, e.g. about 25-35 wt%, e.g. about 30 wt%.Without wishing to be bound by theory, it is believed that a concentration of carbohydrate, e.g. glucose, between about 10 and 50 wt%, e.g. about 10-40 wt%, e.g. about 20-40 wt%, e.g. about 25-35 wt%, e.g. of about 30 wt%, may avoid poor material degradation associated with lower amounts, while avoiding reduction in strength of the material and its ability to retain integrity associated with higher amounts.The composition and / or surgical scaffold, e.g. one or more regions thereof, may further comprise a cellular material, e.g. cells.The cellular material, e.g. the composition, may be provided in the form of a bioink. When a bioink is provided, the scaffold may be cross-linked, such as to provide a stable scaffold and / or increase the mechanical stability of the scaffold. The bioink may further comprise one or more cross-linkers.The cellular material and / or the bioink may comprise adipose-derived mesenchymal cells (adMSCs). Advantageously, adMSCs can be harvested from a patient by a simple, minimally invasive procedure involving liposuction, which is advantageous over other options such as bone marrow harvesting which is far more invasive. Harvesting cells directly from the patient would greatly reduce the risk of rejection of the scaffold after insertion as it would be derived from the patient themselves. adMSC cells can be expanded in vitro before providing or seeding, e.g. printing, undifferentiated cells in the scaffold.The cells may be suspended in a hydrogel, which may be selected from an alginate, gelatin or GelMA hydrogel or a medical grade hydrogel bioink such as Gelrin C™, HyStem®, or the like.55723204-1The cellular material, e.g. cells, may be provided within one or more regions, e.g. layers, of the scaffold.The cellular material, e.g. cells, may be provided within each region of the scaffold. In such instance, the scaffold includes cells within each region, e.g. layer, which may grow to exhibit different mechanical and / or structural properties within each region or layer, so as to mimic an anatomical part of the body, e.g. a bone structure.The cellular material, e.g. cells, may be provided within some, e.g. one or two, regions or layers of the scaffold. In such instance, the scaffold includes cells within some, e.g. one or two, regions or layers, which may grow to exhibit different mechanical and / or structural properties within the associated region(s). Cells may be provided within the scaffold after implantation via migration and growth of the patient’s own cells into the scaffold.The cells may be provided, e.g. printed, within the scaffold during manufacture, e.g. 3D printing thereof.Alternatively, the cells may be provided, e.g. seeded, into the scaffold, e.g. into one or more regions or layers thereof, after manufacture of the scaffold.After seeding, one or more regions, e.g. layers, of the scaffold may be provided with appropriate cues, e.g. osteogenic and / or chondrogenic cues, so as to promote differentiation of the cellular material into respective bone regions.For example, the first region may be provided with chondrocytes in order to promote growth of the cellular material into a cartilage-like structure. The third region may be provided with osteoblasts in order to promote growth of the cellular material into a bone-like structure. The second region may be provided with osteoblasts and / or chondrocytes, e.g. with a mixture of osteoblasts and chondrocytes, in order to promote growth of the cellular material into a subchondral bone-like structure.In addition, or alternatively, the first region may be provided with stem cells, such as mesenchymal stem cells, in order to promote growth of the cellular material into a cartilage-like structure. In some examples, the stem cells, such as mesenchymal stem cells, may differentiate into chondrocytes in the first region. The third region may be provided with stem cells, such as mesenchymal stem cells, in order to promote growth of the cellular material into a bone-like structure. In some examples, the stem cells, such as mesenchymal stem cells, may differentiate into osteoblasts in the third region. The second region may be provided with stem cells, such as mesenchymal stem cells, in order to promote growth of the cellular material into a subchondral bone-like structure. In some examples, the stem cells, such as mesenchymal stem cells, may differentiate into osteoblasts and / or chondrocytes in the second region.The scaffold may be configured for use as a surgical implant or a surgical prosthesis, e.g. as a knee prosthesis. The scaffold may be configured for use as a femoral component and / or as a tibial component of a knee replacement implant.The scaffold may be configured to be weight bearing. The scaffold may be configured for use as an implant and / or prosthesis intended to bear the weight of a patient. Typically, the scaffold may have a compressive strength of about 0.5MPa to about 8MPa, e.g. about 1 MPa to about 6MPa, e.g. about 2MPa to about 5MPa. In some embodiments, the scaffold may have a compressive strength of about 0.5MPa to about 2MPa, about 1 MPa to about 3MPa, about 2MPa to about 6MPa, or about 5MPa to about55723204-18MPa. As the skilled person would appreciate, the compressive strength may be measured using a suitable compression test machine available in the art, for example.The scaffold may be configured to degrade over time in vivo. Typically, the scaffold may be configured to degrade in vivo in about 7 days to about 14 days, about 2 weeks to about 4 weeks, about 2 weeks to about 12 weeks, 4 weeks to about 12 weeks, 4 weeks to about 12 months, about 7 days to about 6 months, e.g. in about 14 days to about 3 months, e.g. in about 4 weeks to about 12 weeks. In some embodiments, the scaffold may be configured to degrade in vivo in about 7-14 days, 14-28 days, 10-12 weeks, 3-6 months, 6-9 months or 9-12 months.The scaffold may be configured to degrade over time in vitro. Typically, the scaffold may be configured to degrade in vitro in about 7 days to about 9 months, e.g. in about 14 days to about 6 months, e.g. in about 2 weeks to about 12 weeks. In some embodiments, the scaffold may be configured to degrade in vivo in about 7-28 days, or 4-12 weeks.It will be appreciated that the preferred degradability parameters may depend on a number of factors, such as the specific composition used to manufacture the scaffold, the physical properties of the scaffold such as pore size, strands size, and porosity, the specific location where the scaffold is to be implanted, whether the cellular material has been printed or seeded within the scaffold, and whether a cell maturation step has been carried out prior to implantation.According to a second aspect, there is provided a surgical scaffold, wherein the surgical scaffold is made from a composition comprising a biocompatible material and a graphene-based material, and wherein the surgical scaffold has a first region having a first set of functional properties, and a second region having a second set of functional properties.The biocompatible material may comprise a polymeric material. The biocompatible material may comprise a biodegradable material. Typically, the biocompatible material may comprise a polymeric material, which may comprise one or more polymers selected from the list consisting of polycaprolactone, polylactic acid, poly(lactic-co-glycolic) acid, polyglycolide, polylactide, polyhydroxobutyrate, chitosan, and hyaluronic acid.Typically, the polymeric material may comprise polycaprolactone (PCL). Advantageously, polycaprolactone is biologically inert and biodegradable and therefore well suited for the present application.The graphene-based material may comprise one or more graphene compounds or graphene derivatives selected from the list consisting of graphene, graphene oxide, reduced graphene oxide, and functionalised graphene materials.Typically, the graphene-based material may comprise reduced graphene oxide (rGO). Advantageously, rGO may help improve the biocompatibility of the scaffold and / or may increase mechanical strength.The surgical scaffold has a first region having a first set of functional properties, and a second region having a second set of functional properties. The surgical scaffold may have a third region having a third set of functional properties.The first region may comprise a first set of functional properties, wherein said first region may comprise one or more components configured to promote growth of cartilagelike structure.55723204-1In one example, the first region may comprise one or more components, wherein said one or more components of the scaffold promote differentiation of cells into chondrocytes. In some examples, said first region of the scaffold may comprise one or more cells. In one example, said one or more cells may comprise stem cells. In some examples, said one or more cells may comprise mesenchymal stem cells; optionally, said mesenchymal stem cells are patient-derived mesenchymal stem cells. In some examples, the one or more cells may comprise chondrocytes. In some examples, the one or more cells may comprise a combination of stem cells and chondrocytes.The second region may comprise a second set of functional properties, wherein said second region may comprise one or more components configured to promote growth of subchondral bone-like structure.In one example, the second region may comprise one or more components, wherein said one or more components of the scaffold promote differentiation of cells into osteoblasts and / or chondrocytes. In some examples, said second region of the scaffold may comprise one or more cells. In one example, said one or more cells may comprise stem cells. In some examples, the one or more cells may comprise mesenchymal stem cells; optionally, said mesenchymal stem cells are patient-derived mesenchymal stem cells. In some examples, the one or more cells may comprise osteoblasts and / or chondrocytes. In some examples, the one or more cells may comprise a combination of stem cells, chondrocytes and osteoblasts.The third region may comprise a third set of functional properties, wherein said third region may comprise one or more components configured to promote growth of bone-like structure.In one example, the third region may comprise one or more components, wherein said one or more components of the scaffold promote differentiation of cells into osteoblasts. In some examples, said third region of the scaffold may comprise one or more cells. In one example, said one or more cells may comprise stem cells. In some examples, the one or more cells may comprise mesenchymal stem cells; optionally, said mesenchymal stem cells are patient-derived mesenchymal stem cells. In some examples, the one or more cells may comprise osteoblasts. In some examples, the one or more cells may comprise a combination of stem cells and osteoblasts.In some examples, the one or more components of each region of the scaffold may be embedded within scaffold (e.g. mixed with rGO prior to printing the scaffold) and / or coated on the surface of the scaffold.As it would be appreciated by one skilled in the art, the scaffold of the present disclosure may be provided with mesenchymal stem cells (e.g. patient-derived MSCs) in vitro. For example, after constructing the scaffold, patient-derived MSCs may be expanded in vitro and seeded onto the scaffold. Without wishing to be bound by theory, the functional properties of each region, and / or the mechanical and / or structural properties, may promote formation of layered tissues comprising bone, subchondral bone and cartilage which mimics the in vivo layered tissue.In examples wherein MSCs are expanded in vitro and seeded onto the scaffold, the MSCs may be expanded for up to about 2 weeks, 3, weeks, 4 weeks, 5 weeks or 6 weeks in culture. In some examples, the MSCs may be expanded and cultured for up to 4 weeks. In some examples, the MSCs may be expanded and cultured for up to 5 weeks. In some examples, the MSCs may be expanded and cultured for up to 6 weeks.55723204-1Each region of the scaffold may have one or more of the following mechanical and / or structural properties: a porosity of in the range of about 60% to about 90%, e.g. about 70% to about 90%; a pore size, e.g. average pore size, in the range of about 200 to about 800 pm; a concentration of graphene-based material, e.g. rGO, in the range of about 0.1% to about 1% by weight, based on the total weight of the scaffold in the first region; polymer strands having a diameter, e.g. average diameter, in the range of about 100-500pm.The scaffold detailed herein may have one or more of the following mechanical and / or structural properties: a porosity of in the range of about 60% to about 90%, e.g. about 70% to about 90%; a pore size, e.g. average pore size, in the range of about 200 to about 800 pm; a concentration of graphene-based material, e.g. rGO, in the range of about 0.03% to about 1 % by weight, e.g. based on the total weight of the scaffold; optionally, in the range of about 0.1 % to about 1% by weight polymer strands having a diameter, e.g. average diameter, in the range of about 100-500pm.The scaffold detailed herein may have one or more of the following mechanical and / or structural properties: a porosity of in the range of about 60% to about 90%, e.g. about 70% to about 90%; a pore size, e.g. average pore size, of about 400 pm; a concentration of graphene-based material, e.g. rGO, of about 0.03% by weight, e.g. based on the total weight of the scaffold; optionally, in the range of about 0.1 % to about 1% by weight; polymer strands having a diameter, e.g. average diameter, of about 400pm.In any of the examples detailed herein, the scaffold may further comprise a carbohydrate, such as glucose. The scaffold may comprise the carbohydrate, such as glucose, at a concentration of at least 10% by weight. In one example, the composition may comprise the carbohydrate, e.g. glucose, at a concentration of at least 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% by weight. The scaffold may comprise the carbohydrate, such as glucose, at a concentration of up to 50% by weight. The scaffold may comprise the carbohydrate, such as glucose, at a concentration of at least 10% and up to 50% by weight. The scaffold may comprise the carbohydrate, such as glucose, at a concentration of at least 20% and up to 50% by weight. The composition may comprise the carbohydrate, e.g. glucose, at a concentration of about 20-40 wt%, e.g. about 25-35 wt%, e.g. about 30 wt%.Without wishing to be bound by theory, it is believed that a concentration of carbohydrate, e.g. glucose, between about 10 and 50 wt%, e.g. about 10-40 wt%, e.g. about 20-40 wt%, e.g. about 25-35 wt%, e.g. of about 30 wt%, may avoid poor material degradation associated with lower amounts, while avoiding reduction in strength of the material and its ability to retain integrity associated with higher amounts.55723204-1In one example, the scaffold may comprise the carbohydrate, such as glucose, at a concentration of about 10%, 20% or 30% by weight. In one example, the scaffold may comprise the carbohydrate, such as glucose, at a concentration of about 30% by weight.In some examples, a monosaccharide particle, such as a glucose particle, may have a diameter in the range of about 30 pm to 80 pm. In some examples, a monosaccharide particle, such as a glucose particle, may have a diameter in the range of about 38 pm - 75 pm. The range of the particle size detailed herein may be advantageous for allowing the monosaccharide, such as glucose, to incorporate into the scaffold and / or for printing of the scaffold.According to a third aspect there is provided a method of preparing a surgical scaffold, the method comprising: providing a composition comprising a biocompatible material and a graphenebased material, additive manufacturing the composition to form the scaffold.The method may comprise: additive manufacturing the composition to form a first region of the scaffold having a first set of mechanical properties and / or structural properties; and additive manufacturing the composition to form a second region of the scaffold having a second set of mechanical properties and / or structural properties.In one example, there is provided a method of preparing a surgical scaffold, such as any of the scaffolds detailed herein, the method comprising: providing a composition comprising a biocompatible material and a graphenebased material; additive manufacturing the composition to form a first region of the scaffold having a first set of mechanical properties and / or structural properties; and additive manufacturing the composition to form a second region of the scaffold having a second set of mechanical properties and / or structural properties; and optionally, additive manufacturing the composition to form a third region of the scaffold having a third set of mechanical properties and / or structural properties.The method may comprise preparing a scaffold according to the first aspect.The composition may be a composition as described in the first aspect.The method may comprise additive manufacturing, e.g. 3D printing, a plurality of regions having different mechanical and / or structural properties.The method may comprise additive manufacturing, e.g. 3D printing, the composition to form a third region of the scaffold having a third set of mechanical properties and / or structural properties.The method may comprise preparing the composition.The method may comprise dissolving the biocompatible material, e.g. polycaprolactone, and the graphene-based material, e.g. rGO, in a solvent, e.g. acetone, to form a solution. The method may comprise providing, e.g. pouring, the solution in a container, and evaporating the solvent, so as to obtain the composition, e.g. a PCL-rGO composition in solid form. The method may comprise providing the composition into suitable form for additive manufacturing, e.g. 3D printing. For example, the composition may be provided as particles, e.g. pellets, or as filaments.55723204-1The method may comprise preparing a plurality of compositions, each composition being tailored for preparing a respective region, e.g. layer, of the scaffold.The method may comprise preparing a first composition suitable for use in forming a / the first region. The first composition may comprise the graphene-based material, e.g. rGO, at a concentration of about 0.1% to about 0.3% by weight.The method may comprise preparing a second composition suitable for use in forming a / the second region. The second composition may comprise the graphenebased material, e.g. rGO, at a concentration of about 0.3% to about 0.6% by weight.The method may comprise preparing a third composition suitable for use in forming a / the third region. The third composition may comprise the graphene-based material, e.g. rGO, at a concentration of about 0.5% to about 0.8% by weight.The composition, e.g. the first, second and / or third composition, may further comprise an additive such as a pharmaceutically active ingredient (API), an antibiotic, a growth factor, an analgesic, or the like. By such provision, a respective portion of the scaffold may act as a drug-eluting scaffold. Examples of additives may include TGF-p, VEGF and BMP-2 for bone and cartilage engineering, or dexamethasone for improved osteogenic differentiation. In some examples, the composition may further comprise a carbohydrate, such as glucose.The method may comprise additive manufacturing, e.g. 3D printing, each region of the scaffold, e.g. the first region and the second region.Typically, the method may comprise sequentially additive manufacturing, e.g. 3D printing, each region, e.g. the first region and second region, and optionally the third region.The method may comprise 3D printing one or more regions, e.g. each region, using a SunP Biomaker 3D printer at about 80°C.The method may comprise printing the composition, e.g. to form one or more regions of the scaffold, in a long “S” shape to create each layer. The method may comprise printing the composition at about 45-90°, typically at about 90°.The method may comprise forming the first region by printing the first composition as strands having a diameter, e.g. average diameter, in the range of about 200-400pm. In one example, the strands may have a diameter, e.g. average diameter, of about 400pm.The method may comprise forming the second region by printing the second composition as strands having a diameter, e.g. average diameter, in the range of about 150-300pm. In one example, the strands may have a diameter, e.g. average diameter, of about 400pm.The method may comprise forming the third region by printing the third composition as strands having a diameter, e.g. average diameter, in the range of about 100-200pm.The method may comprise providing the cellular material, e.g. cells, within the scaffold, during the additive manufacturing of the scaffold. In such instance, the method may comprise co-printing the cellular material with or within the scaffold, e.g. during 3D printing of one or more regions thereof.The method may comprise additive manufacturing, e.g. 3D printing, the composition, using a first print head. The method may comprise printing the cellular55723204-1material using a second print head. Preferably, the first print head and the second print head may be different.The method may comprise providing the cellular material, e.g. cells, within the scaffold, subsequent to the additive manufacturing of the scaffold. In such instance, the method may comprise seeding the cellular material within the scaffold, e.g. within one or more regions thereof.The method may comprise preparing a surgical prosthesis or implant, e.g. a knee implant or component thereof. The method may comprise constructing a femoral component and / or a tibial component of a knee replacement implant.The features described in relation to the first aspect or the second aspect may equally apply in relation the method of the third aspect, and vice versa, are not repeated here merely for reasons of brevity.According to a fourth aspect, there is provided a method of manufacturing a surgical scaffold via additive manufacturing, the method comprising: obtaining an electronic file representing a geometry of a scaffold according to the first aspect or the second aspect; and controlling an additive manufacturing apparatus to manufacture, over one or more additive manufacturing steps, the scaffold according to the geometry specified in the electronic file.According to a fifth aspect, there is provided a computer program comprising computer executable instructions that, when executed by a processor, cause the processor to control an additive manufacturing apparatus to manufacture the scaffold of the first aspect or the second aspect.For the avoidance of doubt, the features described in relation to any aspect may equally apply to any other aspect and are not repeated merely for reasons of brevity.BRIEF DESCRIPTION OF DRAWINGSEmbodiments of the invention are described with reference to the accompanying drawings, in which:Figure 1 shows an illustration of a natural bone structure showing cartilage, subchondral bone and bone;Figure 2 shows a schematic view of a surgical scaffold according to a first embodiment; andFigure 3 shows a schematic view of a surgical scaffold including a cellular material, according to a second embodiment.Figure 4 shows an image of scaffolds printed with two different PCL-rGO compositions.Figure 5 (a- side view; b- top view) shows a layered scaffold showing 200, 300 and 400pm pores.Figure 6 shows load deformation curves for different rGO products in PCL-rGO scaffolds.Figure 7 shows load deformation curves of Graphitene rGO compared to plain PCL.Figure 8 shows load deformation curves for Graphitene rGO for small pore size scaffolds.55723204-1Figure 9 shows stress relaxation curves for PCL-rGO materials.Figure 10 shows cell viability (MTT) results for PCL-GO materials.Figure 11 shows cell viability (MTT) results for PCL-rGO materials.Figure 12 shows mineralisation results for HOS cells in Graphitene PCL-rGO scaffolds.Figure 13 shows sulphated GAGs results for SW1353 cell in Graphitene PCL- rGO scaffolds.Figure 14 shows ADSC differentiation for bone (mineralisation) on PCL- graphitene rGO scaffolds.Figure 15 shows ADSC differentiation for cartilage (sulphated GAG) on PCL- graphitene rGO scaffolds.Figures 16-21 show in vivo histology results for scaffold (rabbits at 3, 26 and 39 weeks post-implantation.Figures 22-28 detail work conducted in relation to exemplary glucose / rGO / PCL scaffolds.Figure 29 show results of MTT (cell viability), Alzirazin red (calcium deposits) and Alcian Blue (sulphated GAG content) assays.Figure 30 [A] PCL-Resin with 0.03% w / v KarbQ, very low concentration of graphene which has led to an improved print quality compared to [B] PCL-Resin on its own.Figure 31 shows cell viability (MTT) for 0.1% Graphitene rGO / PCL and 0.1% KarbQ / PCL. Scaffolds at 7 & 14 days following seeding of SW1353, n=6.Figure 32. Cytotoxicity test on coated scaffolds.DETAILED DESCRIPTION OF DRAWINGSFigure 1 shows an illustration of a natural bone structure 10 showing cartilage 40, subchondral bone 30 and bone 20. Further shown are tidemark 36, calcified cartilage 34 and cement line 32.Figure 2 shows a schematic view of a surgical scaffold generally designated 100 according to a first embodiment. The scaffold 100 generally mimics a natural bone structure, such as natural bone structure 10 of Figure 1 , and has a first region 120 configured to promote growth of a bone-like structure, a second region 130 configured to promote growth of subchondral bone-like structure, and a third region 140 configured to promote growth of a cartilage-like structure.Figure 3 shows a schematic view of a surgical scaffold 200, according to a second embodiment. The surgical scaffold 200 includes a scaffold 250 generally similar to the scaffold structure 100 of Figure 2, but in this embodiment further includes a cellular material 260.Figure 4 shows PCL-rGO scaffold printed with two different PCL-rGO compositions. The upper layer is 0.1 % graphitene by weight and the lower layer is 0.5% graphitene by weight. The scaffold has 400pm strands and 400pm pores.Figure 5 (a- side view; b- top view) shows a 3D printed layered scaffold in PCL- rGO. The scaffold is 10mm square by 12mm high. All strands are 400pm. The bottom layer has 200pm pores, the middle layer 300pm pores and the top layer 400pm pores.Figure 6 shows load deformation curves for 1000pm pore scaffolds of different compositions of PCL-graphitene rGO. a) 0.1% graphitene by weight b) 0.5% graphitene55723204-1by weight c) 1.0% graphitene by weight. These show the smallest deformations for the 0.1 % graphitene by weight for the same compressive load.Figure 7 shows load deformation curves of PCL-Graphitene rGO compared to plain PCL showing that the PCL-Graphitene rGO have smaller levels of deformation than PCL for the same compressive load.Figure 8 shows load deformation curves for Graphitene rGO for small pore size scaffolds (200pm and 400pm) showing the much smaller displacements for the same load for the graphitene PCL-rGO scaffolds when compared to plain PCL of the same pore size.Figure 9 shows stress relaxation curves for PCL-rGO materials.Figure 10 shows murine ADSC cell viability (MTT) results for various PCL-GO materials. The positive control is cells growing in a well plate. All the PCL-GO materials show a reduction in cell viability when compared to the positive control and plain PCL scaffolds.Figure 11 shows murine ADSC cell viability (MTT) results for various PCL-rGO materials. The positive control is cells growing in a well plate. The PCL-rGO materials show similar viability to the positive control and plain PCL scaffolds with the graphitene- rGO having slightly better results and the SG rGO-V20.Figure 12 shows mineralisation results for HOS cells in Graphitene PCL-rGO scaffolds. Mineralised deposits (Alizarin Red) for osteosarcoma (HOS) cells on 200pm & 400pm rGO-PCL scaffolds. Blue bars are positive controls (cells in well plate without scaffolds). Red bars are plain PCL scaffolds. Percentage given is percentage of rGO by weight in the PCL. These results are for staining of the scaffold only - cells growing on the well plate are not included.Figure 13 shows sulphated GAGs results for SW1353 cell in Graphitene PCL- rGO scaffolds. Sulphated GAG deposits (Alcian Blue) for chondrosarcoma (SW1353) cells on 200pm & 400pm rGO-PCL scaffolds. Blue bars are positive controls (cells in well plate without scaffolds). Red bars are plain PCL scaffolds. Percentage given is percentage of rGO by weight in the PCL. These results are for staining of the scaffold only - cells growing on the well plate are not included.Figure 14 shows ADSC differentiation for bone (mineralisation) on PCL- graphitene rGO scaffolds with 200pm and 400pm pores. All scaffolds show good bone mineralisation.Figure 15 shows ADSC differentiation for cartilage (sulphated GAG) on PCL- graphitene rGO scaffolds with 200pm and 400pm pores. For 200pm pores sulphated GAG production is higher than plain PCL.Figure 16 shows in vivo histology results for scaffold at 3 weeks postimplantation. Pseudosynovial lining observed at scaffold interface (1). Fibrous connective tissue with vessels (granulation-like) (2). Early woven bone formation adjacent to scaffold (3). Lamellar bone present further out (maturation) (4). Scaffold stable, some empty spaces from degradation (5). Growth of cartilage cells (6). Host response: pseudosynovium + fibrous tissue (normal) (7).Figure 17 shows in vivo histology results for scaffold at 26 weeks postimplantation. Safranin O staining shows scaffold remnants (1). Bone growth and bone islands well established within scaffold (2). Mix of woven and lamellar bone, lamellar55723204-1predominates (3). Remodelling: transition from immature to mature bone (4). Cartilage growth and organisation (5).Figure 18 shows in vivo histology results for scaffold at 26 weeks postimplantation. Bone growth and bone islands well established within scaffold (1). Mix of woven and lamellar bone, lamellar predominates (2). Remodelling: transition from immature to mature bone (3). Cartilage growth and organisation (4).Figure 19 shows in vivo histology results for scaffold at 39 weeks postimplantation. Extensive lamellar bone infiltration across scaffold (1). Woven bone and cartilage (2). Bone filling all gaps and orienting lamellar tissues (3). Graphene particulates still present but there are signs of degradation (4).Figure 20 shows in vivo histology results for scaffold at 39 weeks postimplantation. Extensive lamellar bone infiltration across scaffold (1). Woven bone and cartilage (2). Cartilage layers cover superficial areas (3).Figure 21 shows in vivo histology results for scaffold at 39 weeks postimplantation.Figure 22 shows solvent casting of 30 % glucose in 0.1 % rGO / PCL.Figure 23 shows an exemplary scaffold - 30% glucose / 0.1 % rGO / PCL scaffolds, 06mm x 1.8mm height.Figure 24 shows an exemplary scaffold - 30% glucose / 0.1 % rGO / PCL scaffolds, 014mm x 1.8mm height.Figure 25 shows reduction in mass for 30 % glucose / 0.1 % rGO / PCL against control material across seven 24-hour Pseudomonas lipase treatments.Figure 26 shows cell viability (MTT) of chondrocytes at 8 days following seeding on scaffolds with or without glucose (n=5). *: P < 0.05 (independent T-test, equal variances assumed (Levene significance > 0.05).Figure 27 shows cell viability (MTT) of chondrocytes at 28 days following seeding on scaffolds with or without glucose (n=6).Figure 28 shows cell viability (MTT) at 7 days following MSC seeding on either 0.1% Graphitene rGO / BOCSci PCL scaffolds (control) or 30% glucose / 0.1 % Graphitene rGO / BOCSci PCL scaffolds, soaked in water over a range of times (n=1).Figure 28 shows absorbance of MTT, alcian blue and alizarin red assay of human mesenchymal stem cells matured for 19 days in layered scaffolds in a stirred bioreactor.Figure 29 show results of MTT (cell viability), Alzirazin red (calcium deposits) and Alcian Blue (sulphated GAG content) assays.Figure 30 [A] PCL-Resin with 0.03% w / v KarbQ, very low concentration of graphene which has led to an improved print quality compared to [B] PCL-Resin on its own.Figure 31 shows cell viability (MTT) for 0.1 % Graphitene rGO / PCL and 0.1% KarbQ / PCL. Scaffolds at 7 & 14 days following seeding of SW1353, n=6Figure 32 shows A. MTT readings at 490nm. There is a statistically positive impact of the inclusion of KarbQ for the 10% gelatin coating. No statistical impact is observed for the other concentrations. B. Picture of the coated scaffold after the MTT staining and before the dye resuspension for measurements. The membranes are still stable and homogeneous. C. Pictures of cells on the coated scaffolds at D7.55723204-1EXEMPLARY METHODOLOGY AND RESULTSI. Graphene scaffold 3D printingProducing PCL-rGO materialThe 45kDa molecular weight PCL (704105, Sigma Aldrich, Gillingham, UK) was used with both the Standard Graphene rGO-V20 (Standard Graphene, Ulsan, Republic of Korea) and Graphitene rGO (Graphitene, Scunthorpe, UK). Solvent casting was used to create a homogenous material (film).Solvent casting process:1. A clean glass 100 ml bottle, or a glass universal, should be obtained.2. Graphene product should be weighted directly into the bottle. The exact graphene measured should be noted and used as the basis for the calculation of the mass of PCL to be used.3. The mass of PCL should be 1000x that of the graphene for a 0.1% graphene product e.g. 0.01 g of graphene would be 10 g PCL. The PCL should be weighed in a disposable weigh boat and exact mass noted. For other percentages the amount of PCL should be calculated.4. PCL should then be added CAREFULLY to the graphene in the glass bottle or universal. This should be done by rolling the granules, one at a time, if possible, down the side of the tipped bottle. This is to ensure minimal disturbance of the graphene.5. A clean magnetic stirrer bar should then be carefully added to glass universal using the same technique.6. Acetone sufficient to give a final concentration of 20% PCL / Graphene should then be measured by glass measuring cylinder. This should then be added to the glass bottle or universal, again carefully, by trickling down the side of the tipped bottle. The lid should then be returned to the bottle.7. Mixture should then be stirred at 60°C and 250rpm for at least 2 hours or until fully dissolved8. Solution is poured into a glass Petri dishes and the acetone evaporated overnight in the fume cupboard. Only a thin layer of solution should be put into each dish. For 100ml this is 3 petri dishes.9. Once the acetone has evaporated, and only solid material remains, the material should be removed from the Petri dish, broken up into small pieces and placed in a 100 ml bottle or other air-tight container.0.1%, 0.5% and 1% (by weight) PCL-graphene material was produced. A brittle, homogenous film was produced which could be easily loaded into the heated print head chamber.3D printing of PCL-rGOThe PCL-rGO was used to make 3D printed scaffolds. The PCL-rGO material was printed using a Biomaker2 printer (SunP Biotech, Beijing, China). The BIOMAKER2 settings were altered in a methodical manner to optimise the printing. A manufacturing procedure was defined.55723204-1Using this method different sizes of test scaffold were produced. Strands were printed at 90° rotation in subsequent layers Printing temperature was 80°C, print bed temperature was room temperature,Example 1 - Printing of Scaffold to fit a 24 well plate.These were 14mm in diameter and 2mm high. A 400pm nozzle was used and the line distance was set to 1.4mm (pore size 1000pm). Printing temperature was 80°C, print bed temperature was uncontrolled (room temperature), extrusion speed was 0.5mm3 / s and movement (printhead) speed was 5mm / s.Example 2 - Printing of Scaffold for mechanical compression testing.These were 10mm square and 5mm high. A 400pm nozzle was used and the line distance was set to 1.4mm (pore size 1000pm). movement (printhead) speed was 5mm / s.Different PCL-rGO compositions were printed. The different compositions required different extrusion speeds (Table 1).Table 1 : Extrusion speeds for different PCL-rGO materialsPrinted scaffolds were assessed for accuracy. Light microscope images were used to assess pore size and strand thickness. These showed good results with pore printability above 0.9 (Table 2):Example 3 - Printing of Scaffold with different materials for layered tissuesTo produce the layered tissues containing bone, subchondral bone and cartilage required for joint implants needed scaffolds with different characteristics throughout to provide the correct environments for the different cell types and extracellular matrix (ECM) production.55723204-1Scaffold composition and morphologyThe following characteristics for the scaffolds for layered tissues were selected based on previous work & results, (see Section II & Figure 8 for load bearing results, Section V & Figures 12 & 13 for biocompatibility results)CompositionFrom the compression testing results it was decided that 0.1 %wt Graphitene PCL-rGO was the best material for the load bearing characteristics of the scaffold. From a biocompatibility point of view for bone cells the 0.1 %wt with 400pm pore size was slightly better than the 0.5%wt. For cartilage cells the 0.5%wt was slightly better at both pore sizes.MorphologyNominal 300pm, 350pm and 400pm pore sizes were selected.The scaffolds were designed to be used in bespoke mini-bioreactor with 010mm scaffolds. Each section - bone, subchondral bone and cartilage - was 2mm deep. This gave a scaffold 010mm x 6mm high.Two Geode files were produced using the SunP Biomaker V2 software advanced GCode writer. There was one file for each material.The first file was for the 0.5% PCL-rGO. This contained the design for the cartilage and subchondral bone sections. The second file was for the 0.1% PCL-rGO. This contained the design for the bone section.Scaffold printingThe scaffolds were printed by importing the GCode produced by the SunP Biomaker V2 software advanced GCode writer into the model tab of the original version of the software. This defined the layer height, extrusion speed, print speed and model size.The first chamber for the heated print head was filled with the 0.5% Graphitene rGO-PCL material. The second chamber for the heated printed head was filled with the 0.1 % Graphitene rGO-PCL materialThe first chamber (0.5% PCL-rGO) was placed into the heated print head and pre-heated to 80 °C.The first and second sections were printed together. The first 8 printed layers were printed with a grid pattern with a line distance of 600pm with a layer height of 0.26mm, printhead speed of 3mm / s and extrusion of 0.3 mm3 / s. The second 8 printed layers were printed with a grid pattern with a line distance of 700pm with a layer height of 0.26mm, printhead speed of 3mm / s and extrusion of 0.3 mm3 / s.The first chamber (0.5% PCL-rGO) was removed from the heated print head and the second chamber (0.1% PCL-rGO) was placed into the heated print head and preheated to 80 °C. The print head was returned to the print starting position with a height offset to adjusted for the two sections already printed.The third section was printed. The 8 printed layers were printed with a grid pattern with a line distance of 800pm with a layer height of 0.26mm, printhead speed of 3mm / s and extrusion of 0.3 mm3 / s.55723204-1Figure 4 - Image of scaffolds printed with two different PCL-rGO compositionsExample 4 - Printing of Scaffolds with different pore sizes for layered tissuesTo produce the layered tissues containing bone, subchondral bone and cartilage required for joint implants needed scaffolds with different characteristics throughout to provide the correct environments for the different cell types and extracellular matrix (ECM) production.Scaffold designScaffold composition and morphologyThe following characteristics for the scaffolds for layered tissues were selected based on previous work & results, (see Section II & Figure 8 for load bearing results, Section V & Figures 12-15 for biocompatibility results).From the compression testing results it was decided that 0.1 %wt Graphitene PCL-rGO was the best material for the load bearing characteristics of the scaffold. From a biocompatibility point of view for bone cells the 0.1 %wt with 400pm pore size was slightly better than the 0.5%wt. For cartilage cells the 0.5%wt was slightly better at both pore sizes.The 0.1 %wt rGO was selected for this scaffold.To produce bone a nominal 400pm pore size was selected. To produce cartilage a nominal 200pm pore size was selected.To join the bone and cartilage sections together in the section that would become subchondral bone to was decided to print 300pm pores so that there was a transfer between 200 and 400pm pores.The scaffolds were to be used in bespoke bioreactor with 10mm square sample holders. To have the appropriate depth of scaffold for holder the bone section needed to be 5mm deep, the subchondral bone 3mm deep and the cartilage 4mm deep. This gave a scaffold 10mm x10mm x 12mm.Scaffold printingThe scaffolds were printed by importing the GCode produced by the SunP Biomaker V2 software advanced GCode writer into the model tab of the original version of the software. This defined the layer height, extrusion speed, print speed and model size. Four identical samples were printed for each run of the bioreactor. The chamber of the heated print head was filled with the 0.1% Graphitene rGO-PCL mixture and preheated to 80 °C.All prints were printed at 100% Print speed and 90% Extrusion to overcome an over-extrusion problem seen in the initial print. Twelve samples were printed successfully by this method.Figure 5 - layered scaffold showing 200, 300 and 400pm pores.55723204-1Layered scaffold maturationAn exemplary layered scaffold comprising an area with a pore size of 200 pm in diameter, an area with a pore size of 300 pm in diameter and an area with a pore size of 400 pm in diameter were seeded with cells. The scaffold comprising cells were cultured in a bioreactor and assessed.Human adipose derived stem cellsHuman adipose derived stem cells were expanded in MSC Growth medium 2 (Promocell Gmbh, Germany). Each scaffold was seeded with 1 million cells in total. The scaffolds were placed in a 24 well plate with the bottom surface facing up. The cells (P3) were detached with TrypleE Express., counted, centrifuged and resuspended in medium at a concentration of 500,000 cells / 1 OOpl. 10OpI of cells was pipetted onto the surface of the scaffold and returned to the incubator for 30 minutes. After 30 minutes, the scaffold was turned over with sterile forceps and 10Opl (500,000 cells) was pipetted onto the other side of the scaffold. The scaffolds were returned to the incubator to allow attachment for a further 30 minutes. The scaffolds were then placed into the sample baskets with the 400pm pores facing down.Human osteoblasts and chondrocytesHuman osteoblast and chondrocyte cells were expanded in Osteoblast and Chondrocyte Growth medium respectively (Promocell Gmbh, Germany). Each scaffold was seeded with 1 ,000,000 osteoblast cells and 500,000 chondrocyte cells. The scaffolds were placed in a 24 well plate with the bottom surface (400pm) facing up. The osteoblast cells (P4) were detached with TrypleE Express., counted, centrifuged and resuspended in medium at a concentration of 1 ,000,000 cells / 1 OOpl. 10Opl of cells was pipetted onto the surface of the scaffold and returned to the incubator for 30 minutes. After 30 minutes, the scaffold was turned over with sterile forceps and 10Opl (500,000 cells) of chondrocytes (P4) was pipetted onto the other side of the scaffold. The scaffolds were returned to the incubator to allow attachment for a further 30 minutes. The scaffolds were then placed into the sample baskets with the 400pm pores facing down.ResultsThe scaffolds were removed from the bioreactor and sliced into 4 sections, each containing layers of bone, subchondral bone and cartilage. Each section underwent a separate treatment: MTT assay to assess cell viability, Alcian Blue Staining to measure sulphated GAG content (cartilage), Alizarin red staining to measure calcium deposits (bone) and fixation in formalin (4% for 1 hour) for further characterization.MTT AssayThe MTT across the four scaffolds had a mean absorbance of 0.22, significantly higher than an empty well with a measured absorbance of 0.05 (Figure 29).Alizarin Red AssayThe scaffolds were imaged with a Motic Light microscope prior to elution of the stain and significant staining of ECM was visualised (not shown) The mean absorbance of the eluted dye was 0.99 (Figure 29).55723204-1Alcian Blue AssayThe scaffolds were imaged with a Motic Light microscope prior to elution of the stain and significant staining of ECM was visualised (not shown). The mean absorbance of the eluted dye was 0.58 (Figure 29).II. Graphene scaffold characterisationThe PCL-rGO scaffolds that had been printed were tested to define their mechanical properties.MethodologyScaffoldsScaffolds were printed for testing. Initial samples that were 10mm square in area and 5mm deep were printed. It was decided to use a 1000pm pore size as this was the most porous scaffold that was likely to be used and so the least material giving the weakest scaffold. The scaffold was printed with the 400pm nozzle as using this nozzle had given the most consistent printing results.A second run of tests used 200pm and 400pm pores with the 400pm nozzle and the Graphitene rGO at 0.1 % and 0.5%wt. These were the porosities that had been selected for the layered tissue scaffolds.The scaffolds were printed as per the methods given above (section I Example 2) but in summary the PCL-rGO material was prepared by solvent casting with acetone. Once cast the material was broken up into small pieces and placed into the heated printhead chamber. The BIOMAKER2 was set with the appropriate printing settings. The shape of the scaffold was defined in the BIOMAKER2 software.The composition of the scaffolds was varied as follows.Table 3: List of characteristics of different scaffolds printed for compression testing55723204-1Loading requirementsA literature search on the normal loads and stresses seen in the knee had been carried out. These figures were used to generate testing protocols.For the initial tests the maximum stress required was set to be 5MPa. With the 10x10mm samples this was a load of 500N. As the behaviour of the material was not known it was decided to reach this load in three steps. The material was first loaded to 100N, then 300N, then 500N. For each load level the loading was cycled between 5N and the set level five times. The loading rate was set at 0.5Hz (1 Hz from unloaded to maximum load) to approach the loading rate seen in gait.For the second set of testing the samples were again loaded to 5MPa (125N) at 0.5Hz (1 Hz from unloaded to maximum load). In addition, stress relaxation characteristics were investigated with a hold at constant displacement for an initial load of 125N.Compression testingCompression testing was carried out on an Instron ElectroPuls E10000.Compression platens were designed and manufactured. They were made in brass which was deemed to be hard enough compared to the PCL.Each sample was placed into the platens in the Instron machine and a tare compression load of 1.0N applied.Samples were loaded as per the predefined protocols. Tests were run in load control. The load was ramped to 60N then cycled through five cycles of 100N to 5N. The load was then ramped to 160N and cycled through five cycles of 300N to 5N. Finally, the load was ramped to 260N and cycled through five cycles of 500N to 5N. Data recorded were time, load and displacement.For the second tests, the protocol was again run in load control, with the sample pre-loaded as previously. The load ramped from 5N to 125N and cycled the loading and unloading 10 times. The final cycle ended at maximum load and the displacement was held for 1 minute to test the time dependent response of the material.AnalysisThe production of stress I strain graphs based on the overall dimensions of the samples was considered. However, unlike a test of a solid material, with the printed scaffolds the variation in dimensions did not necessarily indicate more or less material, it could as easily indicate the same amount of material but variations in printing giving slightly larger or smaller pores within the scaffold. It was therefore felt that load I displacement was the correct way to compare the results between what were nominally exactly the same scaffolds.The whole test was plotted for each sample to enable visualisation of the progress of deformation across the different scaffolds. Then only the final load-unload curve was plotted to more easily compare the response of the different scaffolds. Finally, the slope of the top of the final loading curve (200N to 500N) was used to calculate a scaffold stiffness.55723204-1Results1000pm scaffoldsAll results showed a fairly wide variation in deformation for the same scaffold material. However, the trends were consistent between materials.The Graphitene rGO scaffolds showed much better results than the Standard Graphene rGO scaffolds with lower levels of deformation. This was particularly apparent for the 0.5% and 1.0% results (Figure 6 a, b & c).When compared to pure PCL the 0.1% and 0.5% Graphitene rGO showed improved deformation with the 1.0% having similar deformation. However, the pure PCL had a much wider variation in results (Fig 7).The stiffness of each scaffold showed lower variation than the deformation although again the PCL had the largest range (Table 4). The 0.1% wt rGO had the narrowest range of stiffness.Table 4: Stiffness of 1000 / pm pore 10x10x5mm scaffolds in compression200pm and 400pm scaffoldsThe smaller pored scaffolds showed the same trend in results as the 1000pm ones. The addition of rGO strengthen the scaffolds when compared to plain PCL and the 0.1 % scaffolds were stiffer than the 0.5% scaffolds (Fig 8).The stress relaxation curves were as expected with the stiffer materials showing less stress relaxation (Fig 9).ConclusionsThe compression testing showed that the PCL-rGO scaffolds were capable of weight bearing loads. The addition of PCL-rGO increased the scaffold stiffness but the effect was not linear. The stiffest scaffolds were with 0.1% rGO. The Graphitene product showed better results than the Standard Graphene one.III. Cytotoxicity testing of graphene scaffoldsCytotoxicity testing of the different materials and scaffolds were carried out to understand the response of cells and tissues to them. Testing included both in vitro and in vivo.PCL-GO and PCL-rGO in-vitro testing4.1.1 MaterialsPCL-GO and PCL-rGO materials of different %wt with the different products (Table 6) were prepared by the solvent casting method given above (section I). Plain PCL was also prepared in the same way.Table 6: Compositions of different GO and rGO materials used for in-vitro testing55723204-1These materials were then placed in 24 well plates (0.2g per well) and baked at 90°C to melt them to form a flat disc at the bottom of the well plate.Once the 24 well plates were prepared with PCL-graphene material they were seeded with murine adipose derived stem cells (ADSCs) at 50,000 cells per well. Plates were matured I kept for 7 days before assessment with assays.AssaysFor the cell cytotoxicity testing it was decided to use MTT to assess metabolism. ResultsThe PCL with GO showed variable results (Fig 10). The Graphitene GO showed poor cell viability compared to the positive control (cells in well plate) and pure POL for all concentrations (0.1% to 3.0%). The Standard Graphene GO-V20 showed an OK result at 0.1% but poor viability for higher concentrations. The GOgraphene GO had a very good result at 0.5% which was the same as the controls.The POL with rGO showed better results than the POL with GO (Fig 11). Both rGO products had viability levels close to the controls (cells directly in 24-well plate or pure POL). For both products there appeared to be a small trend of reducing viability with increasing %wt of rGO.ConclusionThe PCL-rGO compositions gave better cell viability as assessed by the MTT assay than the PCL-GO materials.IV. PCL-rGO ISO testingGLR Laboratories Pvt Ltd (Milton Keynes, UK) were engaged to complete cytotoxicity testing of PCL-rGO scaffolds to ISO10993. The testing was carried out on 2 compositions of scaffold - 0.1 %wt and 0.5%wt of Graphitene rGO in 45,000MW PCL.Samples were prepared by printing the PCL-rGO material into solid cylinders 02mm by 10mm in length.In-vitro testingThe in-vitro cytotoxicity testing was carried out to ISO 10993 Part 5. Both Elution and Direct Contact methods were used.The results for the Elution tests were very similar for both the 0.1 %wt and 0.5%wt (data not shown). The cultures treated with the test item extract at different concentrations (30% to 100%) appeared normal without any change in their morphology (grade 0) when compared with the vehicle control. Cells treated with test item extract at55723204-1different concentrations (30% to 100%) exhibited viability greater than 70% (ranging between 96% to 90%). The negative controls showed viability of > 95% and positive controls performed as expected with a viability < 15%.The results for the Direct Contact were very similar for both the 0.1 %wt and 0.5%wt (data not shown). The cultures treated with the test item did not show any cytotoxic response (grade 0) with no detectable zone around or under specimen. Viability greater than 70% was observed in the cultures treated with test item.The conclusions of the tests based upon the results obtained and in line with ISO 10993-5:2009, were that both compositions were non-cytotoxic to Balb / c 3T3 cells.In-vivo testingThe in-vivo cytotoxicity testing was carried out to ISO10993 Part 6. These used subcutaneous implantation in Wistar rats. Both 2 week and 4 week implantation was carried out. The implanted test items were cylinders of material 02mm x10mm.14 day test resultsThese studies were conducted to evaluate the biocompatibility of PCL-rGO version 1 - Composition A and PCL-rGO version 1 Composition B: 0.5% PCL-rGO supplied by Prometheus Regeneration R&D Limited following 2-weeks subcutaneous implantation in male Wistar rats.There were 12 samples in each study named Test Items and 12 Negative Control (High-density polyethylene (HDPE)) all implanted on subcutaneous dorsal areas of rats.The end point examination was 15 days after initial implantation of Test Item / Negative Control when animals were euthanized in compliance with OECD, FDA and ISO / IEC regulations.• No mortality (first level of outcome) in both reports• No general health issues (second level of outcome) e.g. no weight loss; no illness; no sign of toxicity in both reports• Pathological examination: (third level of outcome): o Both specimens in both studies were encapsulated by thin transparent to opaque white layer of tissue with Test Item showing black colour and Negative Control non specified colour (white?) o The average difference obtained from the biological responses of Test Item and Negative Control were respectively 5.05 for Report 1 (0.1% PCL-rGO) and 4.08 for Report 2 (0.5% PCL-rGO). o According to the table below, Test Item generated a slight reaction with both compositions A and B55723204-1ConclusionThese two reports showed slight reaction to both PCL rGO samples (0.1 % and 0.5%) which support biocompatibility of the scaffold material.28 day test resultsThere were 12 samples in each study named Test Items and 12 Negative Control (High-density polyethylene (HDPE)) all implanted on subcutaneous dorsal areas of the rats.The end point examination was 4 weeks after initial implantation of Test Item / Negative Control when animals were euthanized in compliance with OECD, FDA and ISO / IEC regulations.• No mortality (first level of outcome) in both reports• No general health issues (second level of outcome) e.g., no weight loss; no illness; no sign of toxicity in both reports. Actually, there was weight gain for all rats.• Both specimens in both studies were encapsulated by thin transparent to opaque white layer of tissue with Test Item showing black colour and Negative Control non specified colour (white)• The average difference obtained from the biological responses of Test Item and Negative Control were respectively 3.48 for Study 001 (0.1 % PCL-rGO) and 5.67 for Study 008 (0.5% PCL-rGO)• According to the table below, Test Item generated a slight reaction with both compositions A and B• It is important to notice there were no traumatic necrosis, no foreign debris, no fatty infiltrates and there was neovascularisationAfter 28 days, the response between Test Item and Negative Control are similar in all sites regarding spread of polymorphonuclear leukocytes (PN) and lymphocytes (L). However, it seems that overall number of polymorphonuclear leukocytes (PN) and lymphocytes (L) may have decreased which is correlated to the biological responses of Test Item and Negative Control calculated 5.05 at 14 days and 3.48 at 28 days for composition A and less obvious for composition B 4.08 at 14 days and 5.67 at 28 days. In another words polymorphonuclear leukocytes (PN) are still present to release enzymes on sites but appeared to have done their work quicker with composition A (0.1%) compared to B (0.5%).The report conclusion confirmed minimal number of polymorphonuclear cells, minimal to mild number of lymphocytes, multinucleated giant cells and mild to moderate number of macrophages. At 14 days the conclusion was minimal polymorphonuclear cells, moderate to severe lymphocytes, macrophages and multinucleated giant cells. ConclusionThese two reports showed slight reaction to both PCL-rGO samples (0.1% and 0.5%) like the previous reports at 14 days observed the same, which support relative biocompatibility of the scaffold material.Overall histologic reaction has reduced in particular for composition A between 14 and 28 days.Regarding the scaffold biodegradability, the 4-week studies seem to confirm previous 14 days studies results. The macrophagic reaction was similar for 0.5% PCL55723204-1rGO and 0.1 % PCL rGO after 4 weeks, which was not so obvious at 14 days (there was difference in macrophage numbers between the two compositions). It was a key point that giant cells were numerically more present with our Test Item whatever the composition A or B than the Negative Tests. This probably means that Test Item requires bigger cells than macrophages to phagocyte the fibres as macrophages are equally present in both Test Item and Negative Control whereas giant cells are almost inexistent in Negative Control.V. PCL-rGO scaffold biocompatibilityMethodologyThe testing was carried out in 96 well plates.Scaffold preparationPCL-rGO scaffolds were printed as large sheets (30mm x 30mm) (six layers thick) at a depth of 1.6mm and a tissue punch was used to punch out 6mm discs.Scaffolds were printed using PCL-rGO made with Graphitene rGO. The rGO was selected based on mechanical testing results (data not shown). Samples were plain PCL, 0.1 %wt rGO and 0.5%wt rGO. Scaffolds were printed with 200 and 400pm pores.Cell preparation and seedingRobust cell lines were chosen. For bone the osteosarcoma (HOS) cells were used and for cartilage the chondrosarcoma (SW1353) cells.The printed scaffolds were sterilised with 70% Isopropanol and then rinsed with sterile Phosphate buffered saline (PBS) three times. Each scaffold (well) was seeded with 20,000 cells (density of 62,500 cells / cm2). The HOS cells were incubated mineralisation media (complete DM EM, B-glycerophosphate, ascorbic acid, dexamethasone) for the ECM assay (n=6). The SW1353 cells were incubated in growth media (Complete DMEM:F12) for the ECM assay (n=6).Positive controls of HOS or SW1353 on tissue culture treated plastic were also seeded for the ECM assay.MaturationOnce the scaffolds had been seeded with cells and appropriate media added the 96 well plates were kept in an incubator at 37°C in 5% CO2. Media was changed every 2 or 3 days. Scaffolds were matured for 13 day.Assays and analysisOn day 12 an Alizarin red assay (Sigma Aldrich, Gillingham, UK) to measure mineralised calcium deposits, indicating bone like ECM production was completed on the HOS cells. An Alcian blue assay (Sigma Aldrich, Gillingham, UK) to measure sulphated glycosaminoglycans (GAG) deposits, indicating cartilage like ECM production was completed on SW1353 cells.These assays were chosen to give quantitative results that would allow easy comparison across the different test samples.The results of the plate reader (absorbance) were plotted for each assay using boxplots.Bone (HOS) cellsThe mineralised deposits (Alizarin Red) on all scaffolds showed an increase over the control of no scaffold (cells in well plate) (Figure 12). For the 200pm pores size the rGO55723204-1appeared to reduce mineralisation over the plain PCL scaffold but this was not seen for the 400pm pore size. There was little difference between the 0.1 % and 0.5% rGO scaffolds with a possible weak trend that the 0.5% had lower mineralisation.Cartilage (SW1353) cellsThe sulphated GAG deposits (Alcian Blue) showed a small increase over the control of no scaffold (cells in well plate) (Figure 13) on all scaffolds apart from the plain PCL 400pm pore size. However all results were low values. The deposits for the 200pm pores size appeared to be slightly higher than the 400pm pores size. The rGO appeared to slightly increase sulphate GAG deposits over the plain PCL scaffold. There was a trend that increasing rGO % increased sulphated GAG deposits.VI. Differentiation of stem cells on PCL-rGO scaffoldsTo test the ability of the scaffolds to promote differentiation to osteoblast and chondrocytes a 96-well plate experiment was set up.Scaffold printingThe scaffolds were printed with a 400pm nozzle and punched out using a 06mm tissue punch to fit 96-well plates.PCL-rGO scaffolds were printed as large sheets (30mm x 30mm) at a depth of 1.6mm.Scaffolds were printed in PCL and 0.1% and 0.5%wt Graphitene rGO-PCL with both 200pm or 400pm pore sizes.Scaffold seedingScaffolds were seeded with human adipose derived stem cells (C12977, Promocell Gmbh, Heidelberg, Germany). Cells were seeded at 2x105for promoted differentiation to chondrocytes and 1x105for promoted differentiation to osteoblasts.MaturationInitially scaffolds were incubated with growth medium (MSC Growth Medium 2, Promocell Gmbh) for 2 days. They were then incubated with differentiation medium (chondrogenic or osteogenic differentiation medium, Promocell Gmbh). Scaffolds were incubated for 21 days.ResultsPCL-rGO scaffolds (200pm and 400pm pores, 0.1% and 0.5%rGO) were shown to enable the differentiation of ADSCs. After 21 days there was good cell viability and both bone and cartilage ECM production was seen. All scaffolds gave good bone ECM deposits (Figure 14). For cartilage the rGO scaffolds were better than plain PCL for 200pm but not 400pm where they were the same (Figure 15). For cartilage, there was a dramatic improvement of ECM deposition on scaffolds compared to on tissue culture plastic alone (Figure 15).VII. Degradation following osteochondral implantation in vivo1. Objective55723204-1In vivo degradation of an exemplary 3D-printed PCL-rGO scaffold (polycaprolactone + 0.1 wt% reduced graphene oxide) implanted in a rabbit osteochondral defect model over 3, 26, and 39 weeks.2. Study DesignModel: New Zealand White rabbits (male, skeletally mature).Defect: 4 mm x 4 mm in the patellofemoral groove (non-weight-bearing region).Groups:- 3 weeks — > 3 rabbits.- 26 weeks — > 3 rabbits.- 39 weeks — > 3 rabbits + 1 reserve.- 1 DO rabbit = baseline for histology (performed by sponsor).Assessments: Macroscopic examination and first series of MicroCT scans. Histology deferred to sponsor.3. Surgical and Post-Operative ProcedureStandard aseptic parapatellar approach, defect drilled to 4 mm, scaffold “press-fit” flush with cartilage.Prophylactic analgesics, antibiotics (trimethoprim-sulfa) and anti-inflammatory (meloxicam) were used.Rabbits were monitored for recovery, wound healing, pain, lameness, infection, and body weight.4. Key Observations4.1. ClinicalCommon transient findings: erythema, swelling, scabbing, mild lameness — consistent with normal post-arthrotomy recovery.Two transient infections (Staphylococcus epidermidis and Enterococcus faecal is) linked to self-licking, not implant-related.Weight loss —10—13% during the first 3 weeks; recovery and steady weight gain thereafter.4.2. Macroscopic findingsTimepoint: Integration & Tissue Response; particulates and other Findings.3 weeks: Implants visible, partially or fully covered by white / transparent tissue; well- integrated; normal stiffness. No cartilage damage.55723204-126 weeks: Implants integrated, slight depressions in 5 / 6 sites. Black particulates in all joints (mainly post synovial membrane). Local redness; no lymph-node changes.39 weeks: Implants fully integrated, smooth surface, normal stiffness. Minimal black particulates in 6 / 8 knees, mainly synovium & ligaments very rare in the back of knees. One enlarged lymph node; no cartilage damage.Deviations:One rabbit omitted atipamezole reversal: no impact on results.Wounds rinsed (no iodine) - justified.Post-mortem X-rays omitted (implants not radio-opaque).— > None affected data integrity.5. Conclusions• Stable integration of all PCL-rGO scaffolds up to 39 weeks.• Signs of degradation at 26-39 weeks.• No adverse tissue reactions or cartilage damage observed.• The study demonstrates the exemplary 3D-printed PCL-graphene scaffold detailed herein is biocompatible and integrates well in vivo, with controlled surface degradation starting between 6-9 months.• Findings support further histological and biomechanical evaluation in large- animal models prior to clinical translation.VIII. Assessment of bone and cartilage regeneration in graphene-enhanced PCL scaffolds using micro-CT1 ■ OverviewStudy aim: Assess bone and cartilage regeneration in graphene-enhanced PCL scaffolds (polycaprolactone + 0.1 wt% reduced graphene oxide) using micro-CT at 3-, 26-, and 39-weeks post implantation.Samples: 18 cylindrical osteochondral defects (4 mm x 4 mm) in rabbit distal femurs.Objective: Quantify bone ingrowth, scaffold stability, and microstructural evolution.2. MethodologyWhole samples scanned at voxel sizes between 4 pm and 20 pm depending on size. Large data sets (up to 30 GB) cropped to isolate the region of interest (ROI) .Analysis: Gaussian blur to reduce noise — > thresholding — > binarization — > bone volume quantification.Key parameters: Bone volume, trabecular thickness, pore volume, connectivity density.3. Results by Time Point55723204-1• Week 3: Early bone formation, small bone islands bridging pores, woven bone and early cartilage surface.• Week 26: Substantial ingrowth, scaffold well filled, some variability, cartilage formation at surface, micro-movements noted on the top of the sample confirming non full fitting constructs• Week 39: Complete bone filling in three samples, lamellar bone continuous with cortex, stable scaffold.4. ObservationsMechanical stability: despite partial press-fitting bone outgrowth provided fixation.• Graphene effect: stimulated cell orientation, and osteo- / chondrogenesis.• Histology confirms: Woven bone (3 weeks) superficial layers^ lamellar bone (39 weeks) deep layers.• Variability due to scan resolution, defect size, and local mechanical loading.5. Quantitative Summary• Bone volume increases with time; highest at 39 weeks.• Bone density increases and stabilizes at lamellar stage.• Porosity decreases progressively, indicating scaffold filling and degradations.• Variability limits statistical significance between weeks6. Conclusions• 100% biocompatibility, no infection or rejection.• Early bone formation and cartilage surface sealing confirmed.• Strong mechanical fixation and continuity with native bone.• Graphene-based scaffold demonstrated superior regenerative potential.• The graphene-based scaffold shows spontaneous osteo- and chondrogenesis without cell seeding.• Promising as a seedless bone filler and osteochondral graft with strong mechanical integrity and rapid healing.IX. Summary of in vivo histology results for rGO / PCL scaffold post implantation1. Week 3 (Early Stage)• The scaffold area shows a pseudosynovium lining at the interface, similar to what would be seen around an implant in humans.• Beneath this layer, there is fibrous connective tissue with vessels, resembling early granulation tissue.• New woven bone is forming adjacent to this region, characterized by irregular nuclei orientation at the superficial layers of the scaffolds.• Further out, lamellar bone is observed, indicating maturation.• Some scaffold spaces appear empty, reflecting early degradation as soon as 3 weeks.• Overall, by 3 weeks the scaffold is well integrated, immobile, and supports early bone tissue ingrowth.55723204-12. Week 26 (Intermediate Stage)• Safranin O staining reveals persistent scaffold remnants and bone growth into the matrix.• Bone is predominantly lamellar, indicating maturation compared with week 3.• Some areas still contain woven bone.• Islands of bone appear within the scaffold, suggesting infiltration and colonization of the scaffold structure.• Differentiation between woven and lamellar bone is more evident: woven bone has irregular nuclei, while lamellar bone is mature and structured.• Overall, the scaffold is being actively remodelled, with increasing amounts of organized bone.3. Week 39 (Late Stage)• More extensive lamellar bone infiltration across the scaffold, both centrally and peripherally.• Presence of woven bone transitioning to lamellar bone, with areas of endochondral ossification (cartilage converting into bone).• Cartilage formation is noted at the edges and on the surface, appearing as new, immature cartilage layers covering parts of the scaffold.• Some specimens suggest fracture-callus-like tissue at loose edges, consistent with ongoing adaptive remodelling at the top of the construct emphasising the lack of full press fitting.• Cutting artefacts are visible: scaffold degradation and bone fragmentation during sectioning make some regions appear empty, though bone is likely present.• By 39 weeks, the construct supports active bone regeneration, with regions of cartilage overlay and remodelling consistent with stable integration.X. Degradation and viability effects of glucose inclusion in reduced graphene oxide / polycaprolactone scaffolds1. IntroductionThe purpose of this work was to investigate the effects of integration of glucose into an rGO / PCL scaffold material, both in terms of its effects on the degradation of the scaffold, and also the effects on cells themselves. Glucose is an important metabolic substrate for cells and may have a complex relationship with chondrocytes in the development of cartilage (Hollander and Zeng, 2019). The effects on chondrocytes of the incorporation of glucose into scaffold material was of particular interest in this work.2. Methodology2.1. Manufacture of 30 % glucose / 0.1 % rGO / PCL materialD-(+)-glucose (Sigma Aldrich, Gillingham, Dorset, UK) was graded by grinding with a mortar and pestle, then filtering through two sieves with 75 pm and 38 pm holes respectively, resulting glucose particles in the range of 38 pm - 75 pm. It was decided55723204-1that an appropriate value of glucose in the material was 30 % by weight. This was deemed appropriate given that initial trial experiments using lower percentages showed poor material degradation, and there were concerns that the use of higher percentages may reduce the strength of the material, and its ability to retain integrity. This graded glucose was then used to produce a 30 % glucose / 0.1 % rGO / PCL material using a solvent casting process.In order to carry out the solvent casting process, appropriate amounts of glucose and 45 kDa - 50 kDa PCL (BOC Sciences, Shirley, NY, UK or Sigma Aldrich, Gillingham, Dorset, UK) were weighed into a 100 ml glass bottle. Separately, the rGO and acetone were measured into a glass vial. The mix of acetone and rGO was then poured into the bottle containing the PCL and glucose. A magnetic stirrer bar was then added to the bottle, the lid placed on the bottle, and the contents were then mixed using a heated magnetic stirrer plate at 60 °C and 250 rpm for around 2.5 hours, by which point it appeared homogenous. The bottle was then removed from the heat source, and the contents poured into three 10 cm diameter glass Petri dishes, with approximately similar volumes of liquid in each dish, before being left overnight in a fume cupboard to allow the acetone to evaporate, leaving only the final 30 % glucose / 0.1 % rGO / PCL material (Figure 22). This material was then removed from the Petri dishes and broken up into smaller pieces, before being stored in an airtight container until used for printing. 0.1 % rGO / PCL material was solvent cast in the same manner as the glucose-containing material.2.2. Printing of scaffoldsThe 30 % glucose / 0.1 % rGO / PCL material was printed using a Biomaker 2 bioprinter (SunP, Cherry Hill, NJ, USA) at a temperature of 120 °C, sufficient to melt the PCL but not the glucose. A 400 pm nozzle was used, with print speed and extrusion settings being manually altered for each print to account for variation in extrusion. Several different scaffold designs were printed, depending on the specific experiment, but all had 400 pm diameter pores and 400 pm strands and were uniform throughout. Printing was carried out in an identical manner for the non-glucose material, except that the printhead temperature was lowered to 80 °C.2.3. Accelerated degradation experimentTo compare degradation of scaffolds with and without glucose, Pseudomonas lipase was used to create accelerated degradation. 4 mm diameter cylindrical scaffolds of 4 mm height were printed in both the material containing glucose, and the material without glucose. Due to the small mass of these scaffolds, three scaffolds of the same material were combined for each sample. Each set of three scaffolds were place in 7 ml bijou containers. Pseudomonas lipase solution (Sigma Aldrich, Gillingham, Dorset, UK) at 4.25 units / ml in deionised water, and 2 ml of this was added to each of the bijous. After 24 hours, the lipase was removed, the scaffolds washed three times in deionised water and then left to dry for at least 24 hours. These were then weighed. Following this, they were again placed within bijous with 2 ml of fresh lipase solution for another 24 hours. This whole process was repeated 7 times.2.4. Cell testing55723204-1Two experiments were performed with cells to understand their interactions with the glucose-containing materials produced:(A) Chondrocytes seeded on scaffolds still containing 30 % glucose.(B) Mesenchymal stem cells seeded on which glucose has been washed out by placement in water for periods of up to 14 days.2.4.1. Preparation and seeding of scaffoldsFor experiment A, 6 mm diameter scaffolds were printed (Figure 23), and then sterilised. For this sterilisation process, the scaffolds were placed in 100 % ethanol overnight, before the ethanol was removed, and the scaffolds were washed three times with deionised water before being allowed to dry in the airflow of a microbiological safety cabinet. 100 % ethanol is known to be less effective at sterilisation than the use of 70 % ethanol (Mcdonnell and Russell, 1999). Therefore, given the likely reduction in efficiency of the ethanol sterilisation process, the scaffolds were also subject to UV sterilisation following this. For this, the scaffolds, lying flat within individual wells of a 24-well plate, were subjected to UV in the safety cabinet for 30 minutes. Following this, the scaffolds were turned over with sterile tweezers and were then subjected to another 30 minutes of UV. The reason for this change in process from the one outlined for experiment 1 . was that a process including prolonged immersion would dissolve at least some of the glucose out the scaffolds, which was not desired for this experiment.Human primary articular chondrocytes (Promocell, Heidelberg, Germany) were seeded on these 6 mm diameter scaffolds, placed in an adherent 96-well plate, at 20,000 cells in 50 pL of complete Promocell chondrocyte growth medium (Promocell, Heidelberg, Germany). The cells were allowed to adhere for one hour before a further 150 pL of the medium was added to each well containing a scaffold. The plates were then returned to the incubator. Two to three times a week, the medium was replaced with 200 pL fresh complete Promocell chondrocyte growth medium. After 8 days, five scaffolds of both the glucose-containing material and the control material were subjected to MTT assay to assess viability. After four weeks the remaining scaffolds were assayed: six of both the glucose-containing scaffolds and the control scaffolds underwent MTT assay.For experiment B, 14 mm diameter scaffolds were printed from 30 % glucose / 0.1 % rGO / PCL (Figure 24).These scaffolds were then placed in deionised water for 1 , 4,7 or 14 days. For control scaffolds, scaffolds were printed using the same design, but of 0.1 % Graphitene rGO / PCL only, with no glucose. These latter scaffolds were not soaked in water at all. Following completion of this washing process, the scaffolds were then sterilised by placement in 70 % ethanol solution, before being left for several hours, and then washed three times with sterile water. The scaffolds were then dried in a sterile airflow prior to cell seeding. The scaffolds were then placed flat within individual wells of a non-adherent 24-well plate.57,000 human adipose tissue mesenchymal stem cells (hMSCs) (Promocell, Heidelberg, Germany) were seeded per scaffold, in a 100 pL volume of mesenchymal stem cell growth medium (Promocell, Heidelberg, Germany) were) seeded evenly over the surface55723204-1of the scaffold. The plate was then placed in the incubator for one hour to allow cells to attach to the scaffold. Following this, 1 ml of the growth medium was added to each well of the plate, and the plate was then returned to the incubator. Over the course of the experiment, the medium was refreshed 2-3 times per week, until the analysis point at four weeks following cell seeding.2.4.2. MTT assayFor 6 mm diameter scaffolds, the assay was carried out in wells of a 96-well plate, and for 14 mm diameter scaffolds, it was carried out in wells of a 24-well plate.Thiazolyl Blue Tetrazolium Bromide (MTT) (Sigma Aldrich, Gillingham, Dorset, UK) was prepared to 10 mM in pH 7.4 phosphate buffered saline (PBS) (pH 7.4), and this solution was then sterile filtered. Where a 96-well plate was used, 50 pL added to the medium within the well, whereas for a 24-well plate, 500 pL was used per well. The samples were then 37 °C for 4 hours with the MTT. Following this time period, all liquid was removed from the well and DMSO (500pl for 24-well plate or 200pl for 96-well plate) was added to each well The contents of each well were then mixed thoroughly by pipetting up and down until an even colour is achieved and all crystals were dissolved. 100 pl of the resulting liquid was from each well to a 96-well plate well. Absorbance was then measured with a Multiskan GO plate reader (Thermofisher, Waltham, MA, USA) at 540 nm.3. Results3.1. Assessment of relative material degradationHigher mass losses were seen in the 30 % glucose scaffolds as opposed to the materials containing only rGO as a PCL additive (Figure 25). After 24 hours in lipase, the 0.1 % rGO / PCL scaffolds (n=4) had reduced to an average of 94.9 % of their initial weight (standard deviation (SD) =0.2 %), whereas the scaffolds containing 30 % glucose had fallen to 78.7 % of their initial weight (SD = 1.2 %). By 168 hours in lipase, the nonglucose scaffolds were an average of 71.6 % of their initial weight (SD = 0.5 %) whereas the glucose containing scaffolds were an average of 47.5 % of their initial weight (SD = 0.8 %).3.2. Chondrocytes on glucose-containing scaffoldsChondrocyte viability at 8 days post-seeding was found to be significantly higher on the 30 % glucose scaffold group that the control Figure 26 (average absorbance of 0.2778 versus average absorbance of 0.2010 for control scaffolds). However, there was no corresponding effect from the 30 % glucose scaffolds at 28 days post-seeding (Figure 27).3.3. Washed glucose scaffoldsMTT assay results are given in Figure 28 at 28 days following seeding of hMSCs on washed glucose scaffolds (n=1). It is possible that the use of 14-day washed scaffolds improves numbers of viable cells, given that the absorbance at 540 nm was 2.7168 for the 14-day soaked scaffold, as opposed to 1.3295 for the control scaffold (no glucose),55723204-1and 1.0168 for the 1-day soaked scaffold. This could be due to surface modification being produced by the leaching out of the glucose from the scaffold strands.4. Conclusions• The inclusion of 30 % glucose into an rGO / PCL material enhanced the degradation of the material during an enzyme-based accelerated degradation experiment.• There was an improvement in cell viability at 8 days post seeding for chondrocytes seeded on a 30 % glucose / 0.1 % rGO / PCL scaffold.• There is some evidence that washing glucose out of a 30 % glucose / 0.1 % rGO / PCL scaffold may improve the viability of cells seeded on the scaffold.XI. Graphene-based ScaffoldAn exemplary Karbohm™ Using a pseudomonase lipase assay, a steady reduction in mass across the scaffold materials was found. The degradation rate of the 0.1 % KarbOhm was the same as the 0.1 % rGO material.Hydrogel KarbOhm™ scaffoldThe objective is to formulate a cytocompatible, stable hydrogel in which KarbQ can be dispersed and cross-linked.First, the solutions are prepared in dl water and cast into petri dishes in an even 2mm thick layer. The gels are left to stabilise 5mn in the fridge before being transferred to a fume hood in an air-tight glass container with glutaraldehyde at the bottom. The samples are elevated on glass tube to not be in direct contact with the chemical and are left to be cross-linked by vapour overnight. This first cross-linking stage is aimed at the gelatin.Secondly, the next day, samples are left to vent for 5mn to get rid of excess glutaraldehyde and are then submerged in 0.5mM FeCh for 2h. This second crosslinking step is aimed at the KarbQ sheets.Once the gels are formed, there stability at 37°C over a week was investigated, as a minimum stability is necessary after implantation in vivo. Gels are weighed and measured at DO, D3, D5 and D7, DO being the day of completion of the two cross linking steps.The degradation test showed that for bulk gels, the gelatin only cross-links properly at the surface of the gel, on less than 0.3mm, showing that the cross-linking protocol may be improper for implantation purposes. The gels with added collagen IV stayed stable over time, which can be explained by the fact that collagen stability is not temperature dependant, and collagen stability is ensured once the initial gelation has taken place (Fig.6). No negative nor positive impact of KarbQ on the gel degradation has been shown so far. To avoid running into cross-linking problems, it has been decided to investigate using the hydrogels as a coating for the developed scaffolds.The solutions are prepared as precedently and 3D-printed biocompatible resin scaffolds are dipped into the hydrogel, put in the fridge to set quickly and avoid running of the excess gelatin to obtain an even coating.The gel-coated scaffolds are then put through the same double cross-linking pipeline.The gel coating obtained is uniform and stable for weeks in the fridge or in the incubator. Swelling and degradation tests on the coated scaffolds are being performed. The cytotoxicity of the gel and coated scaffolds were then evaluated to conclude on a potential positive or negative effect of KarbQ.55723204-1The coated scaffolds do not seem to have any negative impact on the cytoviability. Furthermore, the addition of KarbQ seems to have a statistically positive impact on the cell viability for the 10% gelatin. These results, added to the clear stability of the coating over time in the incubator, demonstrate that a thin coating does perfectly cross-link and could be a good alternative to bulk hydrogels. It could even lead to an improved cytoviability on the scaffolds, as well as a better cell adherence. (Figure 32)It will be understood that the present embodiments are provided by way of example only, and that various modifications can be made to the present embodiments without departing from the scope of the invention.ReferencesHollander, J.M. and Zeng, L. (2019) ‘The Emerging Role of Glucose Metabolism in Cartilage Development’, Current Osteoporosis Reports, 17(2), pp. 59-69. Available at: https: / / doi.Org / 10.1007 / S11914-019-00506-0.Mcdonnell, G. and Russell, A.D. (1999) ‘Antiseptics and Disinfectants: Activity, Action, and Resistance’, CUN. MICROBIOL REV., 12.55723204-1

Claims

CLAIMS:1 . A surgical scaffold made from a composition comprising a biocompatible material and a graphene-based material, wherein the graphene-based material comprises or consists of graphene, reduced graphene oxide (rGO) and / or functionalised graphene materials.

2. The surgical scaffold according to claim 1 , wherein the graphene-based material comprises or consists of reduced graphene oxide (rGO).

3. The surgical scaffold according to claim 1 , wherein the graphene-based material comprises or consists of graphene or functionalised graphene.

4. The surgical scaffold according to any of the preceding claims, wherein the surgical scaffold has a first region having a first set of mechanical properties and / or structural properties, and a second region having a second set of mechanical properties and / or structural properties.

5. The surgical scaffold according to claim 4, wherein the first region is configured to promote growth of a cartilage-like structure and / or the second region is configured to promote growth of a subchondral bone-like structure.

6. The surgical scaffold according to any of claims 4-5, wherein the scaffold comprises a third region configured to promote growth of a bone-like structure.

7. The surgical scaffold according to any of the preceding claims, wherein the composition, optionally the first, second and / or third region(s), comprises a bioink.

8. The surgical scaffold according to any of the preceding claims, wherein the composition, optionally the first, second and / or third region(s) is provided in the form of a bioink.

9. The surgical scaffold according to claim 7 or 8, wherein the bioink comprises a hydrogel, an alginate, gelatin or GelMA hydrogel, a medical grade hydrogel bioink, or a combination thereof.

10. The surgical scaffold according to any of the preceding claims, wherein the scaffold or the bioink comprises a cellular material.

11. The surgical scaffold according to claim 10, wherein the cellular material comprises one or more cells.

12. The surgical scaffold according to claim 11 , wherein the one or more cells comprise or consist of mesenchymal stem cells, adipose-derived mesenchymal cells, and / or cells derived therefrom.55723204-113. The surgical scaffold according to any of claims 11-12, wherein the first, second and / or third regions define region(s) for culture, growth, differentiation and / or maintenance of the one or more cells; optionally, wherein the one or more cells of: the first region comprises or consists of chondrocytes; the second region comprises or consists of osteoblasts and / or chondrocytes; and / or the third region comprises or consists of osteoblasts.

14. The surgical scaffold according to any of the preceding claims, wherein the scaffold has one or more properties selected from: a. a pore size in the range of about 100 to about 800 pm; optionally, in the range of about 200 to about 800 pm; b. a concentration of graphene-based material in the range of about 0.05% to about 1% by weight; optionally, a concentration of graphene-based material of about 0.1 % by weight; c. polymer strands having a diameter in the range of about 100 to about 500pm.

15. The surgical scaffold according to any of any of the preceding claims, wherein the first region has one or more properties selected from: a. a pore size in the range of about 100 to about 250 pm; optionally, an average pore size of 200pm; b. a concentration of graphene-based material in the range of about 0.5% to about 0.8% by weight; optionally, in the range of about 0.4% to about 0.6% by weight; further optionally, a concentration of 0.5%, 0.6% or 0.8% by weight; c. polymer strands having a diameter in the range of about 50 to about 200pm.

16. The surgical scaffold according to any of the preceding claims, wherein the second region has one or more properties selected from: a. a pore size in the range of about 100 to about 500 pm; optionally, in the range of about 250 to about 350pm; further optionally, an average pore size of 300pm; b. a concentration of graphene-based material in the range of about 0.2% to about 0.6% by weight; optionally, a concentration of 0.5% by weight; further optionally, a concentration of 0.2% to about 0.3% by weight; c. polymer strands having a diameter in the range of about 150 to about 300pm; optionally, an average diameter in range of about 250 to about 300pm.

17. The surgical scaffold according to any of claims 6-14, wherein the third region has one or more properties selected from: a. a pore size of about 350 to about 500pm; optionally, an average pore size of 400pm; b. a concentration of graphene-based material in the range of about 0.05% to about 0.3% by weight; optionally, a concentration of 0.05%, 0.1 % or 0.15% by weight;55723204-1c. polymer strands having a diameter in the range of about 200 to about 450pm; optionally, an average diameter of 300pm or 400pm.

18. The surgical scaffold according to any of the preceding claims, wherein the biocompatible material comprises one or more polymers selected from the list consisting of: polycaprolactone (PCL), polylactic acid, poly(lactic-co-glycolic) acid, polyglycolide, polylactide, polyhydroxobutyrate, chitosan, and hyaluronic acid.

19. The surgical scaffold according to claim 18, wherein the biocompatible material comprises or consists of polycaprolactone (PCL).

20. The surgical scaffold according to any of the preceding claims, wherein the scaffold comprises one or more extracellular matrix components.

21. The surgical scaffold according to any of the preceding claims, wherein the scaffold has a compressive strength of about 0.5MPa to about 8MPa.

22. The surgical scaffold according to any of the preceding claims, wherein the scaffold is for use as a surgical knee implant or prosthesis.

23. The surgical scaffold according to any of the preceding claims, wherein the scaffold is configured to, in use, degrade over time.

24. The surgical scaffold according to any of the preceding claims, wherein the scaffold is configured to, in use, degrade in vivo in about 7-14 days, 14-28 days, 10-12 weeks, 3-6 months, 6-9 months or 9-12 months.

25. The surgical scaffold according to any of the preceding claims, wherein the composition further comprises a carbohydrate at a concentration between 10% to 50% by weight; optionally, wherein the carbohydrate comprises or consists of glucose.

26. A method of preparing a surgical scaffold according to any of claims 1-25, the method comprising: providing a composition comprising a biocompatible material and a graphenebased material, wherein the graphene-based material comprises or consists of graphene, reduced graphene oxide (rGO) and / or functionalised graphene materials; optionally, wherein the composition further comprises a carbohydrate; further optionally, wherein the carbohydrate comprises or consists of glucose; additive manufacturing the composition to form a first region of the scaffold having a first set of mechanical properties and / or structural properties; optionally, additive manufacturing the composition to form a second region of the scaffold having a second set of mechanical properties and / or structural properties; and optionally, additive manufacturing the composition to form a third region of the scaffold having a third set of mechanical properties and / or structural properties.55723204-1

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