PVA and PCU based materials for cartilage replacement

Inês Ferreira1, Pedro Nolasco1,2 , Ana Paula Serro1,2

1Centro de Química Estrutural (CQE), Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal

2Centro de Investigação Interdisciplinar Egas Moniz, Instituto Superior de Ciências da Saúde Egas Moniz, Monte da

Caparica, Portugal

January, 2021

Abstract

This study aimed to evaluate the potential of hydrogels based on polyvinyl alcohol (PVA) and polycarbonate urethane (PCU) to replace/substitute cartilage tissues. PVA was combined with acrylamide (AAm) due to its good swelling capacity and lubricating properties. The PCU was combined with cellulose acetate (AC) and carbon nanotubes (NTC) due to their excellent mechanical properties. Attempts were also made to improve the mechanical performance of a PVA based hydrogel through reinforcement with printed structures of PCU+AC and PCU+NTC. In addition, the incorporation of drugs, namely the anti-inflammatory diclofenac (DCF), in the best systems was also studied. The results obtained demonstrate that in the case of PVA, the hydrogel 1PVA:0,2AAm achieved mechanical properties similar to those of PVA, with the advantage of increasing the water absorption capacity, maintain or decrease the coefficient of friction of the material and increase the amount of drug released. PCU is mechanically more resistant than PVA. The addition of 15%AC and 2%NTC to PCU improved the mechanical properties of the material, but decreased the water absorption capacity and led to higher friction coefficients. PCU hydrogels containing AC released the highest amount of drug among the PCU-based hydrogels, with a sustained release of approximately 48h. Reinforcement of PVA-based hydrogels with PCU-based fibrous structures obtained by 3D printing did not improve the material's performance, possibly due to adhesion issues. Keywords: Cartilage replacement materials, polymeric materials, polyvinyl alcohol hydrogels, polycarbonate urethane, reinforced hydrogels, drug release.

1. Introduction The articular cartilage (CA) present in the synovial joints is a viscoelastic material with limited regenerative capacity, since it is avascular, aneural, alymphic tissue. It comprises a solid phase consisting essentially of chondrocytes, collagen and proteoglycans, and a fluid phase containing water and dissolved salts. AC is responsible for the distribution of loads in the joints and decrease the friction during movement1,2. The most common joint pathologies are osteoarthritis (OA) and rheumatoid arthritis (RA), characterized by the destruction of AC, causing joint pain, swelling and loss of function 3. In more advanced stages of joint disease, surgery options are considered. However, whenever possible, a more conservative approach is chosen and only the cartilage or the damaged part is replaced and hence the need to produce biomaterials capable of replacing cartilage. 4,5,6,7,8. PVA hydrogels, due to their characteristics (biocompatibility, high water content and good tribological properties) and certain polymeric PCU materials due to their mechanical properties and biocompatibility, appear to be materials with high potential for this type of application 9,10,11. However, there is still a gap in the market for effective cartilage substitutes materials that mimic the natural joint. In order to improve the performance of PVA and PCU, these materials were combined with other compounds and fiber structures obtained by 3D printing. The main goal of this work is to produce PVA/AAm, PCU/AC and PCU/NTC and PVA-based hydrogels reinforced with PCU-based structures that presents adequate properties to substitute the cartilage and with the ability to deliver a desired drug to guarantee a therapeutic function in the post-operatory period.

2. Materials and Methods 2.1. Materials Polyvinyl alcohol (PVA) (Mw = 89000-98000 g / mol), acrylamide (AAm) (Mw = 71.08 g / mol), ammonium persulfate (APS) (Mw = 228.20 g / mol), 2,2'-azobisisobutyronitrile (AIBN), cellulose acetate (AC) (Mn≈30 000 g/mol), diclofenac sodium salt (DCF) and the phosphate-saline buffer (PBS) were obtained from Sigma-Aldrich. Polycarbonate urethane (PCU) PCr ChronoFlex® C 80A was obtained from AdvanSource Biomaterials. Single-walled and double-walled carbon nanotubes (SW and DWNTs - 90wt% 1-4nm, 5-30µm) were obtained from Cheap Tubes Inc. N, N-Dimethylformamide (DMF) was purchased was prom Chem -Lab. Pure acetone and ethanol 99.5% was obtained José Manuel Gomes dos Santos (JMGS). 96% ethanol was obtained from Carlo ERBA and butyl alcohol-Tert (2-Methyl-2-Propanol) from PanReac. The Distilled and deionized water used (DD water) (ρ ≥ 18 MΩ ∙ cm) was obtained using a Milli-Q® water purification system (Integral 3 Millipore - Darmstadt, Germany).

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2.2 Samples Preparation 2.2.1. PVA and PCU based materials PVA hydrogels and PVA hydrogels with different AAm percentages were prepared according to Table 1. Aqueous PVA solutions (15% w/w) were prepared by dissolving the PVA powder DD water at 90°C for 12 hours. During the dissolution process, the solutions were gently agitated. To add the remaining components, the AAm monomers, the APS and the AIBN initiators, the PVA solutions were placed in a water bath at 40°C with magnetic stirring (230 rpm). APS was added in a 3: 1 ratio of the amount of AIBN, whereas the amount of AIBN used was 0,15% that of AAm 12,13. After total solubilization, the PVA and PVA:AAm solutions were carefully poured into borosilicate glass Petri dishes, staying 4 to 6 hours at room temperature, and then subjected to 2 cycles of freezing (16 hours) and thawing (8 hours). After these cycles, they were allowed to dry in a ventilated oven at 55°C for 24 hours to ensure the AAm polymerization in the PVA:AAm hydrogels14,15. Subsequently, all samples were washed in DD water, periodically changed, for 48 hours and were stored in a hydrated state in sealed containers, at room temperature, until they were used. Polymeric solutions of PCU 15% (w/w) were prepared by dissolving the PCU granules in a solution of DMF (100%) and in a solution of pure acetone and DMF (50/50). Different amounts of AC and NTC were also added to these solutions (Table 2), the former was dissolved with magnetic stirring at room temperature for 24 hours, whereas the latter required an ultrasonic bath (VWR) -USC300T, Leuven) with an average duration of 4 hours. Given that the PCU+NTC solution have a high tendency to precipitate, this had to be agitated before use to ensure the dispersion of the NTC 16,17,18,19. The resulting polymeric solutions were poured into borosilicate glass Petri dishes, remaining in the air for 24 hours, and then immersed in DD water to coagulate the solutions by exchanging the solvents. During this process, the DD water was changed twice a day for 3 days. Table 1. PVA/AAm samples composition.

Table 2. PCU, PCU/AC and PCU/NTC samples composition.

2.2.2. Reinforced materials Structures were produced from PCU+15% AC (Reinforcement 1) and PCU+2%NTC (Reinforcement 2) materials in square mesh using a LULZBOT Mini 3D printer (Fargo Additive Manufacturing Equipment 3D, LLC) controlled using the Cura LulzBot software Edition Version 3.6.20. Reinforcement 1 and Reinforcement 2 structures (dimensions of 30x30x1mm) were obtained with a needle with an internal diameter of 0,26 mm, a fiber diameter of 0,2 mm, a fiber spacing of 1 mm and a printing speed of 0,5 mm/s for Reinforcement 1 and 1 mm/s for Reinforcement 2. The line filling directions corresponded to angles of 90 degrees. The entire printing process was done in DD water, to allow the formation of fibers through solvent exchange20. After printing, all reinforcement structures were washed in DD water, changed regularly in order to remove all solvents. Subsequently, the Reinforcement 1 and Reinforcement 2 printed structures were dried on absorbent paper and placed in the center of borosilicate Petri dishes, over which 1PVA:0,2AAm solutions were poured. The reinforced materials obtained were produced following the methods described above.

2.3. Materials Characterization 2.3.1. Mechanical Properties In order to determine the tensile and compression properties of the hydrated hydrogels, a Instron® 5566 Universal

Samples PVA (mg) AAm (mg) APS (mg) AIBN (mg)

PVA 3750 - - -

1PVA:0,2AAm 3750 750 3,375 1,125

1PVA:0,5AAm 3750 1875 8,438 2,813

1PVA:1AAm 3750 3750 16,875 5,625

Samples PCU (mg) AC (mg) NTC (mg)

PCU (50/50 Acetone+DMF) 3750 - -

90% PCU + 10% AC 3750 375 -

85% PCU + 15% AC (Reinforcement 1) 3188 563 -

75% PCU + 25% AC 2813 938 -

70% PCU + 30% AC 2625 1125 -

50% PCU + 50% AC 1875 1875 -

PCU + 1% NTC 3710 - 37,5

PCU +2% NTC (Reinforcement 2) 3680 - 75

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Testing machine from Instron Corporation, with a load cell of 10 kN, controlled through the software Bluehill® 2 was used. The compression samples were cut out using a 11 mm diameter punch cutter. During the tests, the samples were hydrated with DD water at room temperature. A minimum of 5 repetitions per condition were performed with a test speed of 0,1 mm/s and a maximum compressive stress of 8.42 MPa. For PVA, 1PVA:0,2AAm, 1PVA:0,5AAm, 1PVA:1AAm, PCU, PCU+10%AC, PCU+15%AC, PCU+25%AC, PCU+30%AC, 1PVA:0,2AAm+Reinforcemnet 1(PCU+15%AC) and 1PVA:0,2AAm+Reinforcement 2 (PCU+2%NTC) was applied a pre-load of 1 N. For PCU+1%NTC and PCU+2%NTC was applied a pre-load of 6 N. The tensile specimens were cut out from the hydrogels using a dedicated punch cutter (4 mm of width and 20 mm of length). A minimum of 5 repetitions per condition were performed at room temperature with a test speed of 0,1 mm/s.

2.3.2. Swelling and equilibrium water content The pre-washed, hydrated gels were cut into discs of 8 mm in diameter, hydrated for 24h at room temperature and weighted in order to obtain the hydrated weight (mh). Then the samples were dried at 85°C for 20 hours and weighted in order to obtain the dry weight (md). The percental swelling ratio (%TIE), and the equilibrium water content (%TAE), is given by, respectively, the following equations:

%𝑇𝐼𝐸 =𝑚ℎ−𝑚𝑑

𝑚𝑑× 100 (1)

%𝑇𝐴𝐸 =𝑚ℎ−𝑚𝑑

𝑚ℎ× 100 (2)

Where md represents the weight of dried sample and mh represents the weight of the hydrated sample. All material were al characterized in triplicate.

2.3.3. Tribological Properties The friction coefficient of the materials PCU, PCU+15% AC, PCU+2% NTC, PVA and 1PVA:0,2AAm was characterized using an Anton Paar tribometer (TRB3, Version 9.0.12). The tests were carried out in a PBS solution (lubricant medium), at room temperature, in a ball-on-plate configuration, using a 316L stainless steel (SS) ball (6mm diameter) as counter-body (Luis Aparicio SL) 160261). The normal contact forces, of 5 and 10 N, were applied over a total distance of 10 m (cyclic path of 10 mm), at a linear speed of 3.14 cm/s and frequency of 1 Hz. The tests were done in triplicate, for all materials.

2.3.4. Morphology The samples PCU, PCU+15%AC, PCU+2%NTC, PVA, 1PVA:0,2AAm, 1PVA:0,2AAm+Reinforcement 1 and 1PVA:0,2AAm+Reinforcement 2 were analyzed in a Hitachi S-2400 scanning electron microscope (SEM) with thermionic emission at 15 kV. Discs with 11 mm were cut from the samples and subsequently dipped in liquid nitrogen, sectioned in half and after that kept in DD water. The dehydration process used required the sequential drying of the samples with ethanol 70% vol, ethanol 96% vol and ethanol 100% vol, with a duration of 15 minutes, each repeated 3 times. Then, the samples were dried with liquid Tert-butanol at 40°C, for intervals of 15 minutes, each repeated 3 times, while during the last one the samples were cooled to 4°C. After this process, the samples were placed on a metal plate, previously cooled to -20°C, and kept in a low vacuum oven (-0.083 MPa) for 24 hours at 20°C 21. Before SEM observations the samples were coated with a metallic film of gold and palladium with 20 nm of thickness during 60 s in a Quorum Technologies sputter coater and evaporator.

2.3.5. Drug Loading and Release The release profiles of diclofenac from PVA, 1PVA:0,2AAm, PCU, PCU+15%AC and PCU+2%NTC were characterized in triplicate. For this purpose, disks (diameter of 6 mm) were cut, dried for 24 hours in a vacuum at 40°C and then weighed (dry mass). These samples were loaded by immersion in 3 ml of diclofenac solution in phosphate buffer (PBS) (2 mg/ml) for 14 days at 37°C on a shaker (VWR Mini Incubation Shaker) at 180 rpm. After this period, the loaded disks were removed from the drug solution and wiped with absorbent paper, to remove excess solution on the sample surface. The drug was released under static sink conditions and, for that, the loaded discs were immersed in 3 mL of PBS solution and placed at 37oC on an agitator (VWR Mini Incubation Shaker) at 180 rpm. Aliquots of 300µL were collected over time and the volume removed was replaced with fresh PBS solution, until there was no more release of DFC. The concentration of DFC in the release solution for each sample was obtained using a ThermoScientific UV–VIS MultiscanGO spectrophotometer, by measuring the absorbances at a wavelength of 276 nm 22.

3. Results and Discussion 3.1. PVA and PCU based materials 3.1.1. Mechanical Properties Figures 1 and 2 present the results obtained from the mechanical characterization of the studied materials under compression, whereas Figures 3 and 4 present the results obtained in the tensile assays.

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Figure 2. Mechanical properties of PCU and PVA-based materials, obtained from compression stress-strain curves. a) Modulus of elasticity calculated in the 0-5% strain range corresponds to 0-0,05 mm. b) Compressive toughness calculated up to a deformation of 60%. The error bars correspond to ± Standard Deviation.

PVA:AAm hydrogels have a greater deformation compared to PVA (Figure 1 a)), for equal compressive stresses, and these materials have elasticity modulus values that gradually decrease with the increase in the amount of AAm added (Figure 2 a)). The same behavior is observed in the toughness (Figure 2 b)), indicating that the material's ability to absorb energy during the compression process decreases with increasing AAm. Previous studies have already shown that the addition of AAm causes a decrease in the crystallinity of the PVA segments, leading to a decrease in the interactions between their chains. Consequently, there is a decrease in the mechanical properties and an increase in the deformation of these materials13. It is expected that the decrease in crystallinity may also contribute to the decrease in the creep resistance of PVA: AAm materials12. Figure 1 b) shows that the addition of AC gives rise to materials that suffer less deformation, with higher values of elasticity and tenacity modules (Figure 2) than the PCU. There is an optimum concentration of AC, since for values above 15% it is noted decrease in mechanical properties. Tang, C. et al.23, proposed that the semi-rigid structure of the AC could be responsible for increasing the stiffness of PU + AC composite materials. On the other hand, the addition of NTC increases the resistance of the produced materials, since both the modulus of elasticity and the toughness to compression increase with the concentration of NTC (Figure 2). The addition of 1% NTC did not lead to significant improvements in material properties comparing to the PCU (Figure 1), possibly because the concentration used was not sufficient to affect the organization of the PCU. It should be noted the greater dispersion of the values obtained, both in the modulus of elasticity and in the toughness, in the material PCU + 2% NTC (Figure 2). This seems to indicate a greater heterogeneity of the material due to the fact that the increase in the concentration of NTC diminishes their dispersion in the PCU matrix. NTCs are highly polarizable, thus being subject to Van der Waals forces, which, in turn, originate aggregations along the axis of nanotubes17. This effect is more evident when NTCs are dispersed in a polymer matrix, such as PCU, where entropic forces causes the particle to be close together. All materials based on PVA and PCU showed values of modulus of elasticity within the range of values found for the compression of native articular cartilage (0,24-0,85MPa24 and 0,31–0,80MPa25). From the tensile assays, the PVA and 1PVA: 0,2AAm itself showed the best mechanical properties from PVA-based materials (Figure 3 a)). The worst mechanical properties (Figure 4) of the samples with AAm are due to the inhibition

Figure 1. Typical compression stress-strain curves of developed materials. a) PVA and PVA: AAm materials. b) PCU-based materials produced with the addition of different amounts of AC and NTC.

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of the formation of large crystalline regions, thus weakening the general mechanical properties of the materials13. Another factor that may have influenced their properties were the concentration of the initiators in the solution, as they affect the polymerization and the properties of the hydrogels. Increasing the concentration of the initiator will reduce the length of the polymer chain and the elasticity of the gels produced26.

The addition of AC improves the mechanical properties of PCU based materials, but to a limit value between 10-15% of AC (Figure 4). These results seem to indicate that for high concentrations of AC, this compound, with a semi-rigid molecular structure23, will decrease the interactions between the PCU chains, lowering the crosslinking of the material. Therefore, under the effect of the tensile stress, the PCU+AC will be able to organize into a more compact structure and thus it will On the other hand, the tensile behavior of PCU+NTC depends on the concentration of NTC added (Figure 3 b)). The shape of the PCU+2% NTC curve may indicate that the polymer chains in this sample are less mobile and therefore that the material is more rigid and less ductile in the initial deformation phase (Figure 3 b))17. However, with regard to the elasticity modulus, only the value of PCU+2% NTC is within the range of native articular cartilage values (5-25MPa24).

3.1.2. Swelling and equilibrium water content behavior The addition of AAm, as expected, increases both the swelling and the water content of the materials, however this increase is not very significant, probably due to the small amount of AAm added in 1PVA:0,2AAm. AAm is very hydrophilic compound that forms an interpenetrating polymeric network with the capacity to establish hydrogen bonds with water molecules13. Regarding PCU-based materials, they presented lower swelling and water content than PVA material, given the apolar nature of PCU, although the obtained values are much larger than expected. The fact that the PCU-based materials were prepared by exchanging solvents, may explain these high values27. The addition of AC and NTC decreases the swelling and water content, which seems to be in agreement with the greater modulus of elasticity of these materials. Only PVA-based materials with AAm in their composition have values closer to human articular cartilage (TIE≈300% and TAE≈75%28), but in general all materials presented values lower than those of human articular cartilage.

Figure 4. Mechanical properties of PCU and PVA-based materials, obtained from stress-strain tensile curves. a) Modulus of elasticity calculated in the 0-5% strain range. b) Toughness to fracture. The error bars correspond to ± Standard Deviation.

Figure 3. Typical stress-strain tensile curves of developed materials. a) PVA and PVA:AAm materials. b) PCU-based materials with the addition of different amounts of AC and NTC.

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3.1.3 Morphology The PVA and 1PVA hydrogels: 0,2AAm (Figure 5 a) and b)) have a structure with low roughness and no pores, typical of hydrogels produced by CD28. The addition of AAm did not lead to structural changes in the material's surface. All PCU materials developed had a porous structure on their surfaces and cross sections (Figure 5 c) - e)). The appearance of this porous morphology is due to the coagulation method of the studied films, which involved the exchanging the solvent DMF+Acetone for water 27,29. The addition of AC and NTC gave rise to materials with a different surface morphology than PCU. The addition of these compounds may change the organization of the PCU chains, thus changing the morphology of the pores formed.

3.1.4. Tribological properties Figures 6 present the results obtained from the tribological characterization of the studied materials. PVA-based hydrogels have the lowest friction coefficients among all the characterized materials (Figure 6), since they have the greatest swelling capacity and the surfaces with the least roughness (Figure 5)30. At lower forces (F = 5N), it appears that adding AAm lowers the friction coefficient of the material (Figure 6 a)), as expected, since AAm improves its the hydrophilicity12 attracting a greater amount of water molecules from the medium (PBS solution) to the hydrogel surface, thus allowing greater lubrication of the movement and a decrease in the friction coefficient12. The effect of less swelling capacity and greater surface roughness is more prominent in PCU+AC and PCU+NTC materials, since the addition of AC and NTC decreases the swelling capacity of these materials and, in the case of PCU + AC, also increases the size of the pores observed (Figure 5). The increase in the friction coefficient values obtained at 10N (Figure 6 b)) seems to indicate that the increase in the applied force tends to decrease the thickness of the lubricating fluid layer between the two surfaces and a greater influence of the surfaces morphology to the measured friction coefficient value31, 32. Human cartilage has a friction coefficient of 0.19 when tested in PBS against a stainless-steel counter body at F = 10N28, showing that in these conditions the studied materials have values lower or approximate to those of cartilage.

Figure 5. Microstructure of the surface of PVA and PCU based materials produced, observed by SEM: a) PVA surface. b) Surface of the 1PVA: 0,2AAm. c) Surface of the PCU. d) Surface of the PCU+15% AC. e) Surface of the PCU+2% NTC.

Figure 6. Friction coefficient measured in PVA materials, 1PVA: 0,2AAm, PCU, PCU+15% AC and PCU + 2% NTC. a) Test performed under a normal applied force of 5N. b) Test performed under a normal applied force of a normal applied force of 10N. The error bars

correspond to ± Standard Deviation.

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3.2. Reinforced materials 3.2.1. Mechanical properties Figure 7 present the results obtained from the compression and tensile mechanical assays of the reinforced materials. These results show that the inclusion of reinforcement structures printed from PCU+15% AC and PCU+2% NTC into 1PVA:0,2AAm hydrogel did not have the desired effect (Figure 7 a) and b)). The reasons for this poor performance may be related to the dimensions of the printed fibers, the density of fibers in the structures and the adhesion between the hydrogel matrix and the fibers.

3.2.2 Swelling and equilibrium water content behavior The results show that the presence of the reinforcement structures reduces the swelling of the material 1PVA:0,2AAm, more significantly in the material with Reinforcement 2, but has little effect on the equilibrium water content. It is necessary to consider the possible effect of the presence of a rigid structure within the hydrogel matrix, which can constrain its expansion, since both reinforcement materials have higher elasticity modules than the hydrogel matrix.

3.2.3. Morphology The microstructure of the cross sections of the reinforcement materials (1PVA:0,2AAm with reinforcement 1 and 2) shows that the internal structure of the fibers is not homogeneous, as it is possible to notice the existence of a central area of lower material density (Figure 8-white arrows). The results also show a lack of connection previously referred, if we observe the separation that exists at the interface between the fibers and the material matrix (Figure 8-black arrows). This may explain the poor mechanical results of the reinforced hydrogels.

Figure 7. a) Typical compression stress-strain curves of 1PVA: 0,2AAm hydrogels reinforced with 3D printed structures of Reinforcement 1 (PCU+15% AC) and Reinforcement 2 (PCU+2% NTC). b) Typical stress-strain tensile curves for 1PVA:0,2 AAm hydrogels reinforced with 3D printed structures of Reinforcement 1 (PCU+15% AC) and Reinforcement 2 (PCU+2% NTC). The curves of the 1PVA:0,2AAm materials, PCU+15% AC and PCU+2% NTC were also included for comparative purposes.

Figure 8. Reinforcement structures obtained by 3D printing. a) and b) PCU+15% AC (Reinforcement 1). c) and d) PCU+2% NTC (Reinforcement 2). White arrows – Non-homogenous area inside the fibers. Black arrows - Interface between fibers and material matrix.

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3.3 Drug Release Quantification Figure 9 shows the diclofenac release from each of the studied materials along the time. This result shows that the addition of AAm increases slightly the amount of drug loaded (Figure 9) in relation to PVA, which is in accordance with the difference found in the swelling of these materials. However, the affinity between AAm and the drug may also explain not only this slight increase, but also the faster release for the 1PVA:0,2AAm hydrogel. Indeed, the interaction between AAm and the drug may lower its retention in the matrix, speeding up the release of the drug. Among PCU-based hydrogels, PCU+15%AC was the material that showed the highest amount of DCF released, which seems to indicate that there is a greater affinity of this drug for AC. On the other hand, the surface of this material has the largest pores (Figure 5 d)), which may have promoted the entry of the drug into its structure and its connection to the AC33.

4. Conclusions and future work This work shows that the addition of increasing amounts of AAm to PVA hydrogels in general worsens their mechanical properties. However, despite a content of 20% AAm (1PVA:0,2AAm) does not significantly affect the mechanical behavior of PVA, it increases significantly the water absorption capacity. Also, it maintains or decreases the coefficient of friction of the material, depending on the applied load. The high swelling capacity of PVA hydrogels led them to absorb and release a high amount of drug, although the drug was released in a few hours. In PCU-based materials, it was found that the addition of AC and NTC dramatically improved the mechanical properties of the PCU. These materials are also more resistant than PVA hydrogels. However, the effect of AC on mechanical properties is only beneficial for an amount added up to 10-15% of AC, while higher concentrations lead to a maintenance or a decrease of the values obtained. The water absorption capacity of PCU-based materials decreases with the presence of AC and NTC, which corroborated with a higher the friction coefficients than those of PCU, obtained under 5N and 10N. The release of DCF from PCU-based hydrogels was improved with the presence of AC, possibly due to its more porous structure and its affinity with the drug. This hydrogel was the one that presented the best performance in terms of drug release, ensuring a sustained release of the drug for 48 hours. Taking these results into account, despite the fact that the materials 1PVA:0,2AAm, PCU+15%AC and PCU+2%NTC have shown to be the most promising for replacing human articular cartilage, it would be interesting to study the variations in their friction coefficient values in synovial liquid, instead of the PBS solution . Finally, it would also be important to assess the wear of the hydrogels and study the effect of changing the speed and a greater variation in loads in the tests on the tribological behavior. In the case of reinforced hydrogels, to increase the adhesion between the fibers and the hydrogel matrix, one could resort to fiber surface treatments, while to improve the mechanical properties, one could study the effect of the decrease the diameter of the fibers in the contact area between surfaces or the effect of changing the geometry of the fibers, that is, changing the line filling directions in the structure. 5. Ackowledgement This document was written and made publically available as an institutional academic requirement and as a part of the evaluation of the MSc thesis in Bioengineering and Nanosystems of the author at Instituto Superior Técnico. The work described herein was performed at the Centro de Química Estrutural (CQE) of Instituto superior Técnico (Lisbon, Portugal), during the period of February 2020 - January 2021, under the supervision of Professor Ana Paula Valagão Amadeu do Serro and Doctor Pedro Nolasco.

Figure 9. Diclofenac (DCF) cumulative release profiles from the materials: PCU, PCU+15% AC, PCU+2% NTC, PVA and 1PVA:0,2AAm. Error bars correspond to ± Standard Deviation.

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