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(2013)Biocompatibility of Polyurethanes. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013
J. Pritchett (2019)Total Articular Knee Replacement Using Polyurethane
The Journal of Knee Surgery, 33
Y. Marois, R. Guidoin (2013)Biocompatibility of Polyurethanes
K. Athanasopulu, L. Kutuzova, J. Thiel, G. Lorenz, R. Kemkemer (2019)Enhancing the biocompatibility of siliconepolycarbonate urethane based implant materials
Current Directions in Biomedical Engineering, 5
J. Becher, S. Möller, D. Weiss, J. Schiller, M. Schnabelrauch (2010)Synthesis of New Regioselectively Sulfated Hyaluronans for Biomedical Application
Macromolecular Symposia, 296
A. Magnani, S. Lamponi, R. Rappuoli, R. Barbucci (1998)Sulphated hyaluronic acids : a chemical and biological characterisation
Polymer International, 46
(2009)UHMWP/ Hyaluronan Microcomposite Biomaterials
A. Magnani, S. Lamponi, M. Consumi, R. Barbucci (1999)Biological performance of two materials based on sulfated hyaluronic acid and polyurethane
Journal of Materials Chemistry, 9
L. Kutuzova, K. Athanasopulu, Markus Schneider, A. Kandelbauer, G. Lorenz, R. Kemkemer (2018)In vitro bio-stability screening of novel implantable polyurethane elastomers
Current Directions in Biomedical Engineering, 4
DE GRUYTER Current Directions in Biomedical Engineering 2020;6(3): 20203115 1 1 3 1 Kiriaki Athanasopulu , Dimitra Caltzidou , Asuka Sekishita , Larysa Kutuzova , Günter 1 3 1, 2* Lorenz , Masaru Tanaka and Ralf Kemkemer* Sulfated Hyaluronan coating of polyurethane- based implant materials Abstract: Thermoplastic polycarbonate urethane elastomers biostability and physical properties such as strength and (TPCU) are potential implant materials for treating flexibility as well as their tunable mechanical properties they degenerative joint diseases thanks to their adjustable rubber- can maintain their function in well-designed cyclic loaded like properties, their toughness, and their durability. We implants for years. However, unmodified TPCUs often show developed a water-containing high-molecular-weight sulfated non-specific protein adsorption onto the surface leading to hyaluronic acid-coating to improve the interaction of TPCU inflammatory responses. Furthermore, insufficient with the synovial fluid. It is suggested that trapped synovial tribological properties of TPCUs may result in the formation fluid can act as a lubricant that reduces the friction forces and of wear debris particles, inducing harmful reactions or impair thus provides an enhanced abrasion resistance of TPCU the biological environment . To avoid such disadvantages, implants. Aims of this work were (i) the development of a various surface modification techniques have been proposed coating method for novel soft TPCU with high-molecular to alter surface properties without affecting the bulk sulfated hyaluronic acid to increase the biocompatibility and properties [3,4]. For example, functionalization of joint (ii) the in vitro validation of the functionalized TPCUs in cell replacement material surfaces (UHMWPE, TPCU) with culture experiments. native biomolecules such as hyaluronic acid (HA) can improve the bio-compatibility and mimic the natural synovial PURs, surface modification, sulfated hyaluronic joint ensuring abrasion resistance of chondral implants . acid, implant coating However, it is well known that native HA is susceptible to https://doi.org/10.1515/cdbme-2020-3115 fast enzymatic degradation, especially in the inflammatory environment of arthritic cartilage joints . To achieve a hydrogel-coated TPCU surface resistance against enzymatic 1 Introduction degradation we use high-molecular sulfated hyaluronic acid for the surface modification of soft TPCU (Fig.1). We Recent therapeutic approaches for osteoarthritis focus on the performed in vitro biological tests to investigate the deceleration of the disease progression by using minimally responses of chondrocytes on the modified TPCU, suggesting invasive treatment methods before replacing the knee joint by an improved biological activity. endoprostheses as last therapeutic resort [1,2]. Polymers are potentially interesting materials for knee cushion implants supporting physiological issue regeneration or allowing improved implant performance under high mechanical load. Polyurethanes with systematically variable soft and hard segments are commonly used for load-bearing orthopedic Figure 1: Schematic representation of the functionalization with applications like chondral implants. Due to their long-term sulfated hyaluronic acid (sHyal). 2 Materials and Methods ______ 1,2* *Corresponding author: Ralf Kemkemer 1. Applied Chemistry, Reutlingen University, Reutlingen, Germany 2. Max Planck Institute for Medical Research, Heidelberg, Germany 2.1 Materials Ralf.Kemkemer@Reutlingen-University.de 3. Institute for Materials Chemistry and Engineering, Kyushu Sodium hyaluronate (HA; Mw 700 kDa) was purchased from University, Japan Lifecore. Dimethylacetamide (DMAc), Tetrahydrofuran 1 1 3 Kiriaki Athanasopulu , Dimitra Caltzidou , Asuka Sekishita , 1 1 3 Larysa Kutuzova , Günter Lorenz , Masaru Tanaka . Open Access. © 2020 Kiriaki Athanasopulu et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. Kiriaki Athanasopulu et al., Sulfated Hyaluronan coating of polyurethane-based implant materials — 2 (THF), Methanol (MeOH), Acetone, were purchased from sHyal was then precipitated in acetone and after filtrating and ABCR. N,N′-Dicyclohexylcarbodiimide (DDC), bromoacetic washing, the product was dried at 40 C under vacuum. acid, Sulfur trioxide N, N -dimethylformamide complex Afterwards, sHyal was incorporated on the TPCU-BrAc (SO -DMF), Tetrabutylammonium hydroxide (TBA), surface. The polymer films were immersed in a solution of phosphate buffer saline (PBS), Click-iT-EdU Alexa Fluor 10 mg/ml sHyal in PBS (pH 7.4) at 50 °C to reach the 594 imaging kit and the ion exchanger Dowex 50W-X8 were formation of ester bonds between the COO groups of sHyal purchased from Thermo Scientific. and the reactive bromomethyl groups on the polymer surface. After 24 h the sHyal solution was removed and the samples were rinsed thoroughly with PBS. These samples are referred 2.2 Activation of Polyurethane samples to as TPCU-BrAc-sHyal. Before cell experiments, the cast polymer substrates were placed in a multiwell plate and Within the scope of this study, a newly developed sterilized with 70% ethanol. thermoplastic silicone-polycarbonate-urethane (TPCU) sur- rounded by long polydimethylsiloxane chains was used as a base material described in previously [7,8]. For modification 2.4 Cell culture and proliferation assay with sulfated hyaluronic acid (sHyal), the TPCU-elastomers were functionalized with bromoacetic acid (BrAc) by the In vitro tests of sHyal-coated samples were performed with procedure of Magnani et al. [3,4]. Briefly, in a solution of chondrocytes, isolated from porcine cartilage. Chondrocytes TPCU in DMAc (10% w/w), combined with 0.5M DCC, (passage 2) were cultured in a mixture of DMEM/Ham's F-12 bromoacetic solution (40% w/v in DMAc) was added drop- (v/v 2:3) growth medium, supplemented with 0.15 mM wise. After filtration, the activated TPCU (TPCU-BrAc) was Ascorbic acid 2-phosphate sesquimagnesium salt, 10% FCS precipitated in methanol and dried under vacuum. To obtain and 1% PEN/Strep. To determine the proliferation rate, cells thin polymer films for further experiments, a TPCU solution were seeded on the sample surfaces (20.000 cells/cm ) and (10% w/v in THF) poured into a Teflon petri dish and the cultivated for 24 hours under standard cell culture conditions. solvent evaporated under vacuum (500 mbar). For surface EdU staining was performed using the Click-iT-EdU Alexa modification, specimens were cut into cylinders with 3,5 mm Fluor 594 imaging kit according to the manufacturer's diameter and cleaned by 2-Propanol to remove chemical instructions. Images were acquired by a A-Plan 10×, 0.25 residues. Ph1 objective and counting of nuclei was carried out using the Zen Blue image analysis software (Zeiss Axio Observer). 2.3 Sulfation of Hyaluronic acid and 2.5 Characterization methods immobilization on TPCU surface FTIR: ATR-FTIR spectroscopy (Perkin Elmer, Spectrum For the synthesis of high-sulfated hyaluronic acid (sHyal), one) was used to characterize the synthesis of sulfated the SO3/DMF-complex was used as a sulfation agent. To hyaluronan. Measurements were carried out between 4000 dissolve HA in DMF, the native, water-soluble sodium -1 -1 and 700 cm with a resolution of 4 cm . hyaluronate first needed to be converted into the organic Contact angle: Measurements were performed at room soluble tetrabutylammonium hyaluronate (HA-TBA) salt. temperature using a DSA 10 MK2 goniometer (Krüss). Prior Therefore, a sodium hyaluronate solution (0.5 % w/v in to examination samples were dried, 3 µl deionized water deionized water) was mixed with the ion-exchange resin droplets were placed on the surfaces. Each sample was Dowex (25% w/v), which was previously activated with a measured with 10 drops placed at different positions. Young 40% TBA solution. After stirring overnight at room La Place equation was used for sessile drop fitting. temperature, the HA-TBA solution was filtered and Water uptake: The uptake of water was determined by lyophilized. measuring the amount of absorbed water relative to the dry To obtain sHyal with a high degree of sulfation, 4 g of weight of the films after 7 and 14 days of storage in PBS at SO /DMF-complex was added to 100 ml of an HA-TBA 37°C. solution (0.5% w/v in DMF) and stirred under nitrogen purge SEM: The surface morphology of the samples was observed at room temperature overnight. After reaction quenching, by using scanning electron microscopy (Zeiss DSM 962). Prior adding 100 deionized water the pH of the solution was to examination the sHyal polymer films were freeze-dried adjusted with 0.1 M NaOH (pH 9) for obtaining the and sputter-coated with a thin gold layer. corresponding sodium salt of sulfated hyaluronic the acid. Kiriaki Athanasopulu et al., Sulfated Hyaluronan coating of polyurethane-based implant materials — 3 storage in a simulated body fluid (12d and 30d in buffer 3 Results solution at 37°C). The TPCU-BrAc-coated surfaces (Fig. 3 B, E, H) exhibited a rough texture compared to the untreated Before surface modification of the TPCU samples, the samples (Fig. 3 A, D, G) due to the aggregation of bromo- sulfation of HA was investigated by FTIR analysis. Fig. 2 methyl groups after functionalization. The surfaces showed a shows the spectra of unsubstituted hyaluronic acid, tetra- smoothed topography after coating with sHyal. As shown in butylammonium hyaluronate, and sulfated derivative. After Fig. 3, surface restructuring of the TPCU has occurred and ion exchange, the CH and CH valence and deformation 3 2 the differences in surface topography become less obvious vibrations of the TBA are visible in the ranges 3000-2800 1 -1 -1 -1 after two weeks of storage in physiological solution. The cm and 1487-1464 cm . The peaks at 1438cm , 1387 cm -1 untreated hydrophobic TPCU surfaces exhibited a contact and 990-850 cm are assigned to the R-O-SO group in angle of around 105°. sHyal. The decrease in the free primary hydroxyl group band -1 (ν OH) at 3300 cm indicates a high degree of substitution. Figure 4: Average contact angles of TPCU (blue), TPCU-BrAc Figure 2: ATR-FTIR spectra of HA (black), HA-TBA (orange) (green) and TPCU-BrAc-Hyal (grey) depend to storage conditions and HA-SO (red). After modification with BrAc or sHyal, only a slight decrease SEM pictures (Fig. 3 A-C) reveal structural changes of the of contact angle to 95° was observed, implying only a minor surfaces after each individual modification step. The images hydrophilization and wettability effect of TPCU surfaces. in Fig. 3D-I show significant topography differences after This result may suggest that storage of samples in a physiological buffer solution at 37°C leads to a reorganization of the TPCU microdomain structures. The resulting stabilization of the surface modification can be observed in a decreasing the contact angles of 80° for TPCU and 70° for TPCU-BrAc and 65° TPCU-BrAc-sHyal if stored in PBS immediately after modifying the samples (Fig. 4). Compared to the non-modified material (TPCU), the modified polymers show also a significantly higher water uptake nearly saturation after approximately 7 days (Tab. 1). We suggest that in the aqueous environment, the hydrophilic segments of the bulk polymer (aliphatic polycarbonate, urethane) and the functional groups of the sHyal coating increasingly migrate to the surface, which also increases the surface energy and wettability in an aqueous environment. Table 1: Water uptake of the TPCU films after 7, 14 days of storage in PBS at 37 ° C related to the dry weight at day 0. Sample 7d (%) 14d (%) TPCU 0.73±0.03 0.74±0.04 Figure 3: SEM images of the TPCU, TPCU-BrAc and TPCU- TPCU-BrAc 1.52±0.12 1.80±0.11 BrAc-sHyal films directly after functionalization (top), after 12 d (middle) and 30 d (bottom) storage at 37° in physiological buffer. TPCU-BrAc-sHyal 2.22±0.09 2.23±0.10 Kiriaki Athanasopulu et al., Sulfated Hyaluronan coating of polyurethane-based implant materials — 4 To assess cellular responses to the coatings porcine chondrocytes were cultured in direct contact with the modified TPCUs for 24h. Cells adhere and proliferate to varying degrees on the surfaces, as shown by the microscope images in Fig. 5. Cells cultured on TPCU-BrAc surfaces (Fig. 5B) show morphological alterations as well as reduced cell adhesion and proliferation. To quantify the cell division rate of the chondrocytes on the three different samples a proliferation assays were performed (Fig. 6). The microscopic images of the fluorescence-labeled nuclei are shown in Fig. 5. Red labeled are only cell nuclei with EDU intercalations (DNA duplication), while all cell nuclei on the Figure 6: Cell proliferation determined with Click-it EdU Alexa sample are marked blue. Cells on the TPCU-BrAc-sHyal Fluor 594 assay. Error indicators represent the mean and substrates show a significantly increased cell proliferation standard deviation of 10 samples with * p <0.05. rate compared to unmodified or BrAc-treated TPCU as Author Statement shown in Fig. 6. The adhesion of the chondrocytes to the s Hyal-coated samples is also evident according to the phase Research funding: Financial support by BMBF under grand contrast microscope image in Fig. 5. number 01EC1406C (project TOKMIS) is gratefully acknowledged. Conflict of interest: Authors state no conflict of interest. Informed consent: not applicable. Ethical approval: The conducted research is not related to either human or animal use. References  Marois Y, Guidoin R. Biocompatibility of Polyurethanes. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013. Available from: https://www.ncbi.nlm.nih.gov/books/NBK6422/  Pritchett JW. Total Articular Knee Replacement Using Polyurethane. The Journal of Knee Surgery. Mar;33 (3) (2020), 242-246. DOI: 10.1055/s-0039-1677816.  Magnani, A., Lamponi, S., Rappuoli, R. and Barbucci, R. Sulphated hyaluronic acids: a chemical and biological characterisation. Polym. Int., (1998), 46, 225-240  Magnani, A., Lamponi, S., Consumi, M., & Barbucci, R. Biological performance of two materials based on sulfated hyaluronic acid and polyurethane. Journal of Materials Chemistry, (1999), 9(10), 2393-2398  James, S. P., Oldinski, R. (Kurkowski), Zhang, M., & Schwartz, H. UHMWP/ Hyaluronan Microcomposite Biomaterials. UHMWPE Biomaterials Handbook, (2009). Figure 5: Proliferation assay with chondrocytes after 24 h. (A) 259–276. doi:10.1016/b978-0-12-374721-1.00018-3 TPCU, (B) TPCU-BrAc (C) TPCU-BrAc-sHyal. The cells show an  Becher, J., Möller, S., Weiss, D., Schiller, J. and increased proliferation rate on the sHyal coated surface. Red are Schnabelrauch, M. Synthesis of New Regioselectively cell nuclei of the proliferating cells with EDU storage, blue Sulfated Hyaluronans for Biomedical Application. Macromol. marked all cell nuclei. The scale corresponds to 200 μm. Symp. (2010), 296, 446-452  K. Athanasopulu, L. Kutuzova, J. Thiel, G. Lorenz, R. In conclusion, we successfully achieved a chemical grafting Kemkemer. Enhancing the biocompatibility of silicone of sHyal onto the surface of the novel bio-stable polycarbonate urethane-based implant materials. Curr. Dir. thermoplastic polycarbonate-urethane by a straight forward Biomed. Eng. (2019), 5/1, 453–455  Kutuzova, L.; Athanasopulu, K.; Schneider, M.; Kandelbauer, strategy. The TPCU surface was first functionalized with A.; Kemkemer, R.; Lorenz, G. In Vitro Bio-Stability Screening bromoacetic acid, a spacer, bearing bromomethyl groups as of Novel Implantable Polyurethane Elastomers: anchorage sites. Afterwards, sHyal grafted directly on the Morphological Design and Mechanical Aspects. Curr. Dir. TPCU-BrAc surface. In vitro experiments show an enhanced Biomed. Eng. (2018), 4, 535–538 cell proliferation of chondrocytes on the TPCU-BrAc-sHyal surfaces.
Current Directions in Biomedical Engineering – de Gruyter
Published: Sep 1, 2020
Keywords: PURs; surface modification; sulfated hyaluronic acid; implant coating
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