Bioactive polyethylene for cartilage regeneration

Abstract

Described herein is a polymer blend comprising 80 wt. % to 99 wt. % of ultra high molecular weight polyethylene (UHMWPE); and 1 wt. % to 20 wt.% of a polymer of Formula 1. The polymer blend may be used to make a three dimensionally printed article. The polymer blend may be used in the treatment of osteoarthritis by the generation of chondrocytes.

Description

Bioactive Polyethylene for Cartilage RegenerationREFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to Singapore patent application number 10202500364Y with a filing date of 10 February 2025 and titled “3D Printed Bioactive PE for Cartilage Regeneration” and is incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present disclosure relates to bioactive polyethylene (PE) in cartilage regeneration, the formulation required for optimum chondrocyte proliferation and the design of appropriate chondrocyte test pieces for optimum chondrocyte proliferation.BACKGROUND OF THE INVENTION

[0003] Osteoarthritis (OA) is the third most rapidly rising condition associated with disability, just behind diabetes and dementia. Current treatments of OA are shown in Figure (FIG.) 1 and 2. The current clinical gold standard treatment for early to moderate osteoarthritis with cartilage defects is microfracture surgery (FIG. 1 panel (a) and FIG. 2). However, microfracture treatment has drawbacks with up to 38% failure and a 10-year re-operation rate.

[0004] Autografts (for example, Smith & Nephew Trufit plug, Arthrex Osteochondral Autograft Transfer System (OATS)) harvested from non-weight bearing regions of a patient’s knee are ideal cartilage capped bone plugs but they have limited harvestable quantity and cause significant donor site morbidity. Allografts (for example, Zimmer Chondrofix™) do not possess bone regeneration capability and carry infection risks. To reduce infection risks, the grafts are heavily sterilised but this results in degradation of cartilage material.

[0005] Besides allografts, there are also metallic implants such as Hemicap™ (FIG. 1 panel (b)) but these result in erosion of existing healthy cartilage due to large differences in hardness between metal and cartilage tissue. Intra-articular injections are mostly made of organic materials such as hyaluronic acid or collagen which are too weak to withstand abrasive forces in joints, especially knee joints which are also weight-bearing, thus requiring frequent injections every 3 to 12 months.Furthermore, claims of cartilage regeneration using such materials have been poorly verified clinically. Hence, there remains a strong need to develop cartilage implants that have regenerative properties and able to withstand abrasive forces in joints.

[0006] In summary, the use of autografts in osteoarthritis (OA) are limited by harvestable quantity and donor site morbidity. Allografts do not possess cartilage regeneration capability, often fail to integrate, and carry infection risks. The clinical standard operation microfracture does not restore native cartilage and resulted in up to 38% failure rates. Yet, plugs and metallic implants are used extensively in cartilage repair. Hence, there remains a need to develop novel treatment solutions for patients with early osteoarthritis with the goal of improving outcomes.SUMMARY OF THE INVENTION

[0007] In a first aspect, there is provided, a polymer blend comprising 80 wt. % to 99 wt. % of ultra high molecular weight polyethylene (UHMWPE); and 1 wt. % to 20 wt.% of a polymer of Formula 1.

[0008] The polymer of Formula 1 is:L2 is a substituted or unsubstituted divalent C2 to C6 aliphatic moiety, L3 is a substituted or unsubstituted divalent C2 to C10 aliphatic moiety, L4 is a substituted or unsubstituted divalent C2 to C4 aliphatic moiety,A is selected from the group consisting of RGD, AGQWHRVSVRWG (SEQ ID NO: 3, A5G81), hyaluronic acid, and RLGYVVS (SEQ ID NO: 1),n is from 10 to 350, m is such thathas a number average molecular weight from 500 to 7000,p is 0.9 to 0.99, q is 0.01 to 0.1, p + q = 1.

[0009] In an embodiment, L3 is (CH2)a1, where a1 is from 2 to 8, preferably a1 is from 3 to 7, more preferably a1 is from 4 to 6.

[0010] In an embodiment, L4 is -CH2CH=CH-.

[0011] In an embodiment, n is from 50 to 300, preferably n is from 150 to 250, more preferably n is from 170 to 200.

[0012] In an embodiment, wherein L2 is (CH2)a2, where a2 is from 2 to 6, preferably a2 is from 2 to 4, more preferably a2 is 2 or 3.

[0013] In an embodiment, m is such that the number average molecular weight is from 1000 to 5000, preferably m is such that the number average molecular weight is from 2000 to 4000, more preferably m is such that the number average molecular weight is from 3000 to 4000.

[0014] In an embodiment, A is RGD.

[0015] In an embodiment, the polymer blend consists essentially of 80 wt. % to 99 wt. % of UHMWPE; and 1 wt. % to 20 wt.% of the polymer of Formula 1. In an embodiment, the polymer blend consists of 80 wt. % to 99 wt. % of UHMWPE; and 1 wt. % to 20 wt.% of the polymer of Formula 1. In an embodiment, the polymer blend consists essentially of 90 wt. % of UHMWPE; and 10 wt.% of the polymer of Formula 1. In an embodiment, the polymer blend consists of 90 wt. % of UHMWPE; and 10 wt.% of the polymer of Formula 1.

[0016] In a second aspect, there is provided a three-dimension printed article made of the polymer blend according to the first aspect.

[0017] In an embodiment, the article is a U-shaped article to fit a plate well.

[0018] In a third aspect, there is provided a method of producing a three-dimension printed article. The method comprises determining a shape of the article; and three dimensionally printing the article with the polymer blend according to the first aspect.

[0019] In a fourth aspect, the polymer blend according to the first aspect, or the article according to the second aspect for use in therapy. Preferably, for use in thetreatment of osteoarthritis. More preferably, for use in the generation of chondrocytes in the treatment of osteoarthritis.

[0020] In a fifth aspect, there is provided use of the polymer according to the first aspect, or the article according to the second aspect in the manufacture of a medicament for use in in the treatment of osteoarthritis, preferably for use in the generation of chondrocyte in the treatment of osteoarthritis.

[0021] In a sixth aspect, there is provided a method of treating osteoarthritis, the method comprising inserting a three-dimension printed article made of the polymer blend according to the first aspect into a subject to generate chondrocytes to treat osteoarthritis.

[0022] Advantageously, the polymer blend may be used to generate chondrocytes to treat osteoarthritis.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure (FIG.) 1 shows in panel (a) microfracture treatment for early osteoarthritis and in panel (b) a metallic implant “Hemicap”.

[0024] FIG. 2 shows a comparison of problems with existing solutions and the embodiments described herein.

[0025] FIG. 3 shows an embodiment of a sample design.

[0026] FIG. 4 shows the original polyethylene (PE) samples floating in media.

[0027] FIG. 5 shows the design-specific PE samples in media.

[0028] FIG. 6 shows the in vitro chondrocyte proliferation assay on PE5,000-based materials

[0029] FIG. 7 shows the in vitro chondrocyte proliferation assay on PE5,000-RGD and PE5,000-HA at 48 hours (hr) and 96 hr timepoints.

[0030] FIG. 8 shows the in vitro chondrocyte proliferation assay on PE5,000-RGD in different formulations.DETAILED DESCRIPTION OF THE INVENTION

[0031] In the following description, numerous specific details are set forth in order to provide a thorough understanding of various illustrative embodiments of the invention. It will be understood, however, to one skilled in the art, that embodiments of the invention may be practiced without some or all of these specific details.Embodiments described in the context of one of the methods or products are analogously valid for the other methods or products. Similarly, embodiments described in the context of a method are analogously valid for a product, and vice versa.

[0032] Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, where different elements or groups of elements are disclosed, combinations thereof are also disclosed. Where any element of an invention is disclosed as having a plurality of alternatives, examples of that invention in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an invention can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0033] Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invention. Where a value being discussed has inherent limits, for example where a component can be present at a concentration of from 0 to 100%, or where the pH of an aqueous solution can range from 1 to 14, those inherent limits are specifically disclosed. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the invention, as are ranges based thereon.

[0034] The term "alkyl" as used herein is a branched or unbranched saturated monovalent hydrocarbon radical of 1 to 24 carbon atoms (C1 -C24), such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, f-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or moregroups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0035] The subscript following the polymer is the molecular weight of the polymer used and is the number average molecular weight unless otherwise stated. As an example, PE5,000 refers to polyethylene having a number average molecular weight of 5000 and PEG3,400 refers to polyethylene glycol having a number average molecular weight of 3400.

[0036] Described herein is the three dimensional (3D) printing of bioactive PE for application in cartilage regeneration. In vitro chondrocytes assay showed excellent proliferation at 48hr relative to a control of no treatment. By developing 3D-printable tissue-regenerative PEs.ooo brush copolymers which may be personalized to fit a patient’s anatomy, optimum healing outcome can be achieved. The novel acellular materials used for the implants can encourage tissue regeneration in patients without the addition of foreign cells.

[0037] Described herein is the use of bioactive polyethylene (PE) in cartilage regeneration. To study chondrocyte proliferation, specially designed samples made of bioactive PE were prepared as shown in FIG. 3. Bioactive PE5,000 brush copolymers with different oligopeptides and oligosaccharides at various wt % were blended with a base polymer of ultra high molecular weight polyethylene (UHMWPE L3000) and converted into filament form. These filaments were used in the 3D printer to produce uniquely designed samples for testing. It was determined that the PE samples float in the medium and would result in inaccurate results. Hence, the PE samples were designed based on the dimension of an individual well of the culture plate to be used. A tight fit was incorporated into the overall design (FIG. 3) of the sample which enables it to be firmly secured at the bottom of the well without the use of any adhesive. An experiment was also performed over 7 days to validate the design where PE sample was placed in the cell culture plate containing phosphate buffered saline (PBS). This experiment validated the new design as the sample did not move or float without the use of any adhesive. 3D printing parameters used in preparing the samples can be found in Table 1. By comparing FIG. 4 and FIG. 5, it highlights the significance in creating design specific PE samples for proliferation test by suppressing any sample from floating based on the geometry of well plate used.

[0038] In Fig. 6, sample 1 refers to the base PE which acted as the control with no bioactivity. Relative to the control, samples PE5,000-SVVYGLR (SEQ ID NO: 1) (sample 2), PE5,000-Hyaluronic acid (HA) (sample 3), PE5,000-RGD (SEQ ID NO: 2) (sample 4) demonstrated an improvement of 10-20% in proliferation of human chondrocytes while (PE5,000-A5G81) (sample 5) only had a comparable level as that of control over a treatment period of 96 hours. A5G81 is AGQWHRVSVRWG (SEQ ID NO: 3). The amino acids in the peptides may be L-amino acids, D-amino acids, racemic or a combination thereof, preferably the peptide contains only L-amino acids. The amino acid sequences in the peptides are provided from the N-terminal end to the C-terminal end as per convention, however in the polymer as shown below it is the carboxylic acid of the peptide that bonds to the amino terminated polyethylene glycol. These findings are suggestive of the potential cartilage-regenerative property of samples 2 to 4 and their promise for use as cartilage implant materials.HO

[0039] Upon validating the different types of PE brush copolymers, two of the best performing materials, PE5,000-RGD and PE5,000-HA, were selected for in vitro chondrocyte proliferation test at two timepoints, 48 hr and 96 hr, for further validation (FIG. 7). There is about 15-20% increase in proliferation for PE5,000-RGD at 48 hr. At the later 96 hr timepoint, the cells have grown to saturation in the wells and the difference becomes smaller.

[0040] As PE5,000-RGD is superior to PE5,000-HA in cell proliferation at 48 hr, PE5,000-RGD in 5 weight percent (wt. %), 10 wt. % and 20 wt. % formulation with UHMWPE L3000 (a weight average molecular weight of 500 kDa) were selected for dose screening to determine the optimal formulation for chondrocyte proliferation (FIG. 8). The weight percent of the polymer is measured with respect to the final weight of the blended mixture. Relative to the pure UHMWPE, PE5,000-RGD 5% and PE5,000-RGD 10% revealed an increase of 18% and 40% in chondrocytes proliferation respectively. It is notable that, with the increasing dose of bioaddtive (PE5,000-RGD 20%), there is not only no cell improvement but regression to a same level as PE5,000-RGD 5% (18%) was observed. These findings demonstrated that the optimum formulation for chondrocyte proliferation is PE5,000-RGD 10% in UHMWPE. It is noteworthy that chondrocytes produce collagen which forms hyalinecartilage. Hence, it demonstrates the capability of the bioactive PE in cartilage regeneration.

[0041] Experimental Procedure

[0042] Synthesis of NB-PEG has been described in PCT / SG2020 / 050621 (published as W02022093106) and Guo et al. Polym. Chem., 2023, 14, 1743.

[0043] Synthesis of NBPEG3,400NH2PEG 3400

[0044] PEG diamine (MW 3,400) (3.4g, 1mmol) and cis-norbornene-exo-2,3-dicarboxylic anhydride (1 eq.) were added to a 100 ml round bottom flask (rbf), followed by toluene (50 ml). Triethylamine (1 eq.) was added and the mixture stirred under reflux overnight, with a dean stark trap attached for water removal. The resulting solution was evaporated to dryness and dichloromethane (40 ml) was added, followed by 0.1 M HCI (40 ml). The organic layer was extracted and washed with 0.1 M NaOH (50 ml). Another 0.1 M NaOH (50 ml) was added to the aqueous fraction from the acid wash followed by CH2CI2 (30 ml). The organic layer was extracted and combined, washed with saturated NaCI before drying over Na2SO4. The material was evaporated to dryness to give a yellow waxy solid as the product (3.3g, f yield 93%).1H NMR (CD3OD, 500 MHz, 25°C): 5 = 6.33 (s, 2H), 3.64 (m, PEG), 3.56 (s, 4H), 3.18 (m, 2H), 2.71 (s, 2H), 1.45 (d, 1H, J=7Hz), 1.39 (d, 1H, J=10Hz). ESI-MS (M+H+Na)2+: calculated: 1788.047; found: 1788.5 (n=76).

[0045] General Procedure to prepare norborene-PEG-bioactive moiety monomer

[0046] Synthesis of NBPEG3,4ooRGDPEG 3400

[0047] RGD (with 1 carboxylic acid on aspartic acid protected with OMe) (0.0937 g, 0.26 mmol), was dissolved in MeOH (2.5 ml) in a 4 ml vial, in the glovebox.iPr2EtN (91 µl, 0.52 mmol) was added to form the solution (A). HOBt (0.0353 g, 0.26 mmol) and HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) in a 20 ml vial at 40°C, followed by addition of the RGD solution (A), to give the mixture (B). The mixture (B) is then added to NBPEG3,4ooNH2 (0.77g, 0.218 mmol) in a 40 ml vial and stirred at room temperature (r.t.) for 24h. The resultant mixture was then evaporated to dryness and was added to Et20 (50 ml). The Et20 solution was chilled in a freezer for 48 h and decanted. MeOH (5 ml) was added to the residue and filtration was performed. The filtrate was evaporated to dryness followed by dialysis against deionized water and lyophilization to give a pale yellow solid as the product NBPEG3,400RGD (0.8g, yield 95%).1H NMR (D2O, 500 MHz, 25°C): δ = 6.37 (s, 2H, NB), 4.04 (m, RGD), 3.85 (m, RGD), 3.71 (m, PEG), 3.64 (d, 4H, J=2.5Hz, NB), 3.23 (m, RGD), 3.00 (m, RGD), 2.86 (d, 2H, J=1.5Hz), 1.92 (m, RGD), 1.70 (m, RGD), 1.52 (d, 1H, J=10Hz, NB), 1.32 (d, 1H, J=10Hz, NB). ESI-MS (M+H+Na)2+: calculated: 1716.99; found: 1716.0 (n=65).

[0048] NB-PEG3,400-HA and NB-PEG3,400-RLGYVVS were prepared in a similar manner by replacing the RGD carboxylic acid with hyaluronic acid and RLGYVVS carboxylic acid.

[0049] 1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using D2O as solvent for NB-PEG3,400-RLGYVVS macromonomer. 1,2-C6D4Cl2 was used as the solvent for the PE brush copolymer.HO'

[0050] NB-PEG3,400-RLGYVVS (0.8g, yield 95%). 1H NMR (D2O, 500 MHz, 25°C): δ 7.09 (m, SVVYGLR), 6.84 (m, SVVYGLR), 6.37 (s, 2H, NB), 4.62 (m, SVVYGLR), 4.40 (m, SVVYGLR), 4.29 (m, SVVYGLR), 4.12 (m, SVVYGLR), 3.71 (m, PEG), 3.56 (m, 4H, NB), 3.22 (m, 2H, NB), 3.18 (m, SVVYGLR), 3.01-2.94 (m, SVVYGLR), 2.85 (m, 2H, NB), 2.11-1.91 (m, SVVYGLR), 1.65 (m, SVVYGLR), 1.53 (m, SVVYGLR), 1.52 (m, 1H, NB), 1.33 (m, 1H, NB), 0.97-0.89 (m, SVVYGLR) ppm. ESI-MS (M+2H)2+: calculated: 2075.995; found: 2075.2 (n=72).

[0051] NB-PEG3,400-HA

[0052] Hyaluronic acid (HA, molecular weight 3,000-5,000) (100 mg, 0.025 mmol) was dissolved in deionized (DI) water at pH 5.0-5.5 (10 mL, pH adjusted by 1M HCI), and EDC (3.9 mg, 0.025 mmol) and N-hydroxysuccinimide (NHS) (2.9 mg, 0.025 mmol) were added into the HA solution and stirred for 1 h. NBPEG3400NH2 (88.8 mg, 0.025 mmol) was added to the reaction mixture and stirred at room temperature overnight and the reaction was stopped by adding 1M NaOH; the final pH was adjusted to 7-8. The solution was dialyzed against DI water for 48 h using 5,000 Da molecular weight cut off cellulose membrane to remove uncoupled NBPEG3400NH2 and HA. A pale yellow powder (90% yield) was obtained after the freeze-drying process.1H NMR (D2O, 500MHz): δ 6.35 (br t, 2H), 3.69 (s, PEG), 2.00 (s, HA). ESI-MS (M+H+Na)2+: calculated: 2091.12; found: 2090.2 (y=39).

[0053] Synthesis of NB-SA-t PEs.ooo;■ y— K L-\ llNB-SA-t PE5,000

[0054] Succinic acid-terminated polyethylene (MW 5000) (SA-t PEs.ooo) (10 g, 2 mmol) was weighed into a 250 ml round bottom flask (rbf) followed by addition of toluene (120 ml). Hexamethylenediamine (HMDA) (0.2784 g, 2.4 mmol) and triethylamine (0.28 ml, 2 mmol) were added. The mixture was stirred under refluxovernight connected with a Dean Stark trap for water removal. The resultant suspension was cooled and concentrated, followed by addition of MeOH, to give a beige precipitate. The mixture was filtered and the residue was washed with MeOH before dryness, to give SA-t PEs.ooo-HMDA quantitatively.

[0055] SA-t PEs.ooo-HMDA (10.2 g, 2 mmol) was added to a 250 ml rbf followed by addition of cisnorbornene-exo-2,3-dicarboxylic anhydride (0.366 g, 2.2 mmol), toluene (120 ml) and triethylamine (0.28 ml, 2 mmol). The mixture was refluxed overnight with a connected Dean Stark trap for water removal. The resultant suspension was cooled and concentrated, followed by addition of MeOH, to give a beige precipitate. The mixture was filtered and the residue was washed with MeOH before dryness, to give NB-SA-t PEs.ooo macromonomer (9.4g, yield 90%). 1 H NMR (1.2-C6D4CI2, 400 MHz, 120oC): δ = 6.03 (m, 2H, NB), 5.44 (m, 1H, -CH=CH-), 5.24 (m, 1H, -CH=CH-), 3.39 (m, 6H, -CH2- on HMDA), 3.05 (s, 3H, -CH2- on HMDA), 2.41 (m, 5H), 1.95 (m, 3H), 1.22 (m, PE), 0.84 (M, -CH3).

[0056] General ring opening metathesis polymerisation (ROMP) procedure

[0057] Each of the macromonomers were prepared separately and copolymerised in a random manner using a second generation Grubbs’ catalyst (Gll-catalyst). The NB-PEG macromonomer was weighed into a 20 ml vial followed by addition of NB-SA-t PEs.ooo (5 equivalents). 1,2-diclorobenzene (2.4 ml) was added and the mixture stirred at 75 °C till a brown solution was obtained. A solution of the 2ndgeneration Grubbs’ catalyst in 1,2-diclorobenzene (1.25 mol %, 0.05 M) was added and the mixture was stirred for 24 h at 75 °C. Ethyl vinyl ether was added to the mixture followed by MeOH (15 ml) to give a beige precipitate. The mixture was filtered and the residue was washed with water / acetone before dryness. All copolymers were obtained with the isolated yield over 90%. NB-SA-t PE conversion and NB-PEG-peptide incorporation varied. The relative amount of the NB-PEG-peptide or hyaluronic acid to the polyethylene component range from 1 to 10% in the copolymer.HN=< NH NH2HN=(NH2

[0058] PEs.ooo-RGD copolymer1H NMR (1,2-C6D4Cl2, 400 MHz, 120°C): δ = 6.06 (s, unreacted NB-SA-t PE5,000), 5.48 (m, vinyl proton on SA-t PE5,000), 5.30 (m, vinyl protons on SA-t PE5,000), 3.55 (s, PEG), 3.41 (m), 3.27 (s), 3.09 (s), 2.44 (m), 1.93 (m), 1.28 (s, PE), 0.84 (m, 3H, -CH3 on SA-t PE5,000).

[0059] PE5,000–RLGYVVS was synthesized by ring opening metathesis polymerization (ROMP) as above.

[0060] PE5,000–RLGYVV brush copolymer: 1H NMR (1,2-C6D4Cl2, 400 MHz, 120°C): δ 6.06 (s, unreacted NB-SA-t PE5,000), 5.49 (m, vinyl proton on NB-SA-t PE5,000), 5.31 (m, vinyl proton on NB-SA-t PE5,000), 3.55 (s, PEG), 3.41 (m), 3.07 (s), 2.43 (m), 1.93 (m), 1.28 (s), 0.84 (m) ppm.

[0061] Preparation of polymer blend

[0062] The PE-peptide brush copolymers were formulated as “bioadditive” with UHMWPE L3000 (Mitsui Chemicals) as the base polymer to create the formulations [PE-peptide / UHMWPE], 5 wt. %, 10 wt. % and 20 wt. % of the copolymers were added to the UHMWPE with the weight percentage of the bioadditive being with respect to the total weight of the formulation.

[0063] Sample preparation

[0064] Ø2.85mm filament was produced using ThermoScientific Process 11 twin-screw extruder connected to a melt pump fitted with ø3mm nozzle, water bath and spooling unit. Solidworks 2022 was used for 3D modelling of the sample. Ultimaker Cura software was used for parameter selection and slicing of the 3D model while 3D printing of samples was fulfilled using a Ultimaker S5 dual nozzle 3D printer fitted with AA 0.4mm core. The 3D printing parameters are set out in Table 1 below.

[0065] Table 1. 3D printing parametersParameters ValueNozzle size (NS) 0.4mm (or 0.25mm)Print temperature 210 – 225°CBuild plate temperature 60 – 80°CBuild plate type Polypropylene (PP)Adhesion type BrimBrim width 7mmLayer height 0.06 – 0.2mmInitial layer horizontal expansion -0.05 – -0.1mmLine width 80 – 100% of NSInfill density 100%Infill pattern LinesInfill overlap 0.2 – 0.3mmPrint speed (PS) 5 – 15mm / sCooling speed 0 – 30%Minimum layer time > 60sMinimum speed (cooling) = PSLift head EnabledRetraction distance 7 – 9mmRetraction speed 40mm / sFlow 100 – 105%

[0066] In vitro chondrocyte proliferation assay

[0067] Briefly, human chondrocyte cell line (CHON-001) was maintained in complete Dulbecco's Modified Eagle Medium (DMEM) (G418, 10% fetal bovine serum). To study proliferation, 3D printed U-shaped vessels from different PEs.ooo copolymers

[0068] were sterilized in 70% ethanol before placement in the bottom of 24-well tissue culture plates. 50,000 chondrocytes were seeded into wells with U-shaped PEs.ooo vessels in 500pL complete DMEM. Cells were grown for 48 hours or 96 hours in a 37°C incubator at 5% CO2. 48 or 96 hours later, cells were harvested by detachment with 0.05% Trypsin and cell proliferation was assessed with CyQUANT™ Cell Proliferation Assay kit (Invitrogen, Waltham Massuchusetts) according to the manufacturer’s instructions. The readout of cell proliferation was measured using a microplate reader with excitation at 485 nm and emission detection at 530 nm (Tecan, Mannedorf, Switzerland). Kruskal-wallis test was performed as statistical analysis to assess significant differences in cell proliferation effected by different bioactive PEs.ooo copolymers.17INCORPORATED BY REFERENCE (RULE 20.6)

Claims

Claims1. A polymer blend comprising 80 wt. % to 99 wt. % of ultra high molecular weight polyethylene (UHMWPE); and 1 wt. % to 20 wt.% of a polymer of Formula 1,171Formula 1,l_2 is a substituted or unsubstituted divalent C2 to C6 aliphatic moiety,l_3 is a substituted or unsubstituted divalent C2 to C10 aliphatic moiety,l_4 is a substituted or unsubstituted divalent C2 to C4 aliphatic moiety,A is selected from the group consisting of RGD, AGQWHRVSVRWG (SEQ ID NO: 3, A5G81), hyaluronic acid, and RLGYVVS (SEQ ID NO: 1),n is from 10 to 350, m is such thathas a number average molecular weight from 500 to 7000,p is 0.9 to 0.99, q is 0.01 to 0.1, p + q = 1.

2. The polymer blend according to claim 1, wherein l_3 is (CH2)ai, where ai is from 2 to 8, preferably ai is from 3 to 7, more preferably ai is from 4 to 6.

3. The polymer blend according to claim 1 or claim 2, wherein l_4 is -CH2CH=CH-.

4. The polymer blend according to any one of claims 1 to 3, wherein n is from 50 to 300, preferably n is from 150 to 250, more preferably n is from 170 to 200.

5. The polymer blend according to any one of claims 1 to 4, wherein L2 is (CH2)a2, where a2 is from 2 to 6, preferably a2 is from 2 to 4, more preferably a2 is 2 or 3.

6. The polymer blend according to any one of claims 1 to 5, wherein m is such that the number average molecular weight is from 1000 to 5000, preferably m is such that the number average molecular weight is from 2000 to 4000, more preferably m is such that the number average molecular weight is from 3000 to 4000.

7. The polymer blend according to any one of claims 1 to 6, wherein A is RGD.

8. The polymer blend according to any one of claims 1 to 7 consisting essentially of 80 wt. % to 99 wt. % of UHMWPE; and 1 wt. % to 20 wt.% of the polymer of Formula 1, preferably the polymer blend consists of 80 wt. % to 99 wt. % of UHMWPE; and 1 wt. % to 20 wt.% of the polymer of Formula 1.

9. The polymer blend according claim 8 consisting essentially of 90 wt. % of UHMWPE; and 10 wt.% of the polymer of Formula 1.

10. A three-dimension printed article made of the polymer blend according to any one of claims 1 to 9.

11. The article according to claim 10, wherein the article is a U-shaped article to fit a plate well.

12. A method of producing a three-dimension printed article, the method comprising determining a shape of the article; and three dimensionallyprinting the article with the polymer blend according to any one of claims 1 to 9.

13. The polymer blend according to any one of claims 1 to 9, or the article according to claim 10 or claim 11, for use in therapy.

14. The polymer blend or the article according to claim 13 for use in the treatment of osteoarthritis.

15. The polymer blend or the article according to claim 14 for use in the generation of chondrocytes in the treatment of osteoarthritis.

16. Use of the polymer according to any one of claims 1 to 9, or the article according to claim 10 or claim 11 in the manufacture of a medicament for use in in the treatment of osteoarthritis, preferably for use in the generation of chondrocyte in the treatment of osteoarthritis.

17. A method of treating osteoarthritis, the method comprising inserting a three- dimension printed article made of the polymer blend according to any one of claims 1 to 9 into a subject to generate chondrocytes to treat osteoarthritis.

Images