Startseite Long-term stable modifications of silicone elastomer for improved hemocompatibility
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Long-term stable modifications of silicone elastomer for improved hemocompatibility

  • Cécile Boudot EMAIL logo , Sarah Burkhardt und Miriam Haerst
Veröffentlicht/Copyright: 30. September 2016
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Current Directions in Biomedical Engineering
Aus der Zeitschrift Current Directions in Biomedical Engineering Band 2 Heft 1

Abstract

Silicone elastomers are well established in medical engineering and particularly in blood-contacting applications such as catheters and medical tubing. Still, their intrinsic surface properties have potential for improvement. For example, hydrophobicity reduction can be a way to provide better hemocompatibility. In this study, several bulk and surface modifications of silicone elastomers using polyethylene glycol (PEG) were investigated. All modifications induced long-term (2 months), stable wettability of the surface. Moreover, cytotoxicity testing demonstrated their suitability as implant material. Hemocompatibility was investigated through a thrombin generation assay as well as a platelet adhesion study combining an enzymatic assay and a scanning electron microscope analysis. That the hemocompatibility of silicone was considerably improved thanks to the PEG modifications could be shown. The study introduces easily processable, cost-efficient, and long-term stable hydrophilic modifications of silicone elastomer for improved hemocompatibility.

Keywords: cytotoxicity; hemocompatibility; long-term stability; polyethylene glycol; Silicone elastomer; wettability

1 Introduction

Silicone elastomers are of major interest for medical applications due to their high chemical- and physical-resistance properties combined with elasticity. Silicones in which the organic substitutes are methyl groups are the most widely used silicone elastomers [1]. These silicones are also known as PDMS (polydimethylsiloxanes). High chemical stability enables prolonged use with constant properties despite diverse influences such as hygienic reprocessing and watery environment. PDMS is furthermore referred to as nontoxic [1]. This is why it is for example used in medical tubing, catheters, sealing, or grips on medical devices.

Unmodified PDMS surfaces are hydrophobic and tend to generate uncontrolled protein adsorption, which could induce thrombogenic behavior under specific circumstances [2], [3]. Hydrophilic modifications of PDMS using passivating polyethylene glycols (PEGs) seem to be a promising strategy to improve silicone’s biological surface properties. PEG is well known for its excellent antifouling and antithrombogenic properties [2], [3], [4]. However, the use of PEGs is limited since they are prone to thermal and oxidative degradation [5]. Moreover, PEG-modified silicones often suffer from so-called hydrophobic recovery [6]. Easily processable, stable, and cost-effective modifications of PDMS for biomedical applications are still lacking.

The following study aims at using PEG to hydrophilize silicone surfaces for long-term blood-contacting applications.

2 Material and methods

2.1 Silicone modification

Wacker Chemie AG, Germany, has provided medical-grade high-temperature-vulcanizing (HTV) silicone elastomer (Silpuran® UR 9030/60). This PDMS was used in this study as unmodified reference material and as substrate for the surface modifications. Reference samples are designated below as “Sil”. Vulcanization was carried out in a heating press at 200°C, 50 bars for 10 min.

As an example of bulk modification, a commercially available compound containing a chemically connected polyethylene-glycol-siloxane copolymer was used (Elastosil® RT 629, Wacker Chemie AG, Germany). This room-temperature-vulcanizing (RTV) silicone was vulcanized at 120°C for 30 min and is designated below as “RTV+PEG.” To demonstrate the effectiveness of this PDMS-PEG copolymer, additional samples of the reference silicone Silpuran® UR 9030/60 containing the same copolymer as Elastosil® RT 629 have been provided by Wacker Chemie AG, Germany (designated below as “HTV+PEG”).

After preparation, the samples were post cured (200°C, 4 h), thoroughly cleaned, and dried at room temperature for 24 h. The samples not used for silanization were sterilized with hot steam (121°C, 20 min, 2 bars).

Beside the previous bulk modifications, a coating using an organofunctional silane with PEG function (2-[methoxy (polyethylenoxy)6-9 propyl] trimethylsiloxane, ChemPur, Germany) was prepared. The silicone surface was first oxidized with atmospheric pressure plasma (PT-60, Plasmatreat GmbH, Germany). The silane was dissolved in ethanol and acetic acid at a volume ratio of 1:2:50 (acetic acid:silane:ethanol) and incubated on the oxidized silicone for 5 min at room temperature then heated for 1 h at 110°C. This procedure enables covalent binding of the PEG function on the silicone surface. This sample was designated as “Sil-PEG.” In addition, a coating using amino-functional silane (APTES, Genosil® GF93, Wacker Chemie AG, Germany) was used as positive control for cell adhesion studies [7] and a coating with succinic acid functional silane (Genosil® GF20, Wacker Chemie AG, Germany) was used as positive control for the thrombin generation study [8]. The designations of those samples are “Sil-GF93” and “Sil-GF20.”

After coating, the samples were washed for 20 min in an ultrasonic deionized-water bath, dried, and sterilized with hot steam (121°C, 20 min, 2 bars).

2.2 Suitability as implant material

2.2.1 Long-term stable wettability

Contact angle (CA) measurement gives information about the influence of modifications on the silicone samples’ wetting behavior, which was determined with drops of 2 μl of distilled water at a dosing rate of 2 μl/s. The CA was recorded after drop spreading stabilized on the surface (Contact Angle System OCA and SCA20 software, Dataphysics GmbH, Germany).

According to ISO 10993-1, long-term implants remain in the patient’s body for more than 30 days [9]. In this study, the stability of the modifications was tested through storage in simulated body fluid (SBF, produced according to [10]) at 37°C for 2 months. Finally, the wetting behavior of the stored samples was investigated (contact angle measurement) and compared with samples of the same materials that did not undergo SBF storage.

2.2.2 Cytotoxicity

Verifying that the modified silicones do not induce cytotoxic effects is necessary to investigate their suitability as implant material. Cytotoxicity was evaluated according to ISO 10993-5 [9] using human fibroblasts Hs27 (CRL-1643™, ATCC®, UK). Material samples were extracted in cell culture medium (DMEM, 5% FBS, 1% Pen/Strep, 1% Amphotericin B, Merk KGaA, Germany) for 72 h at 37°C at a surface-volume ratio of 1.5 cm2/ml. Subconfluent fibroblasts (5000 cells/cm2 in a 96-well microplate) were then incubated with 200 μl material extracts for 48 h at 37°C. Potential cytotoxic substances can reduce the number and vitality of cells during incubation. For the quantitative evaluation of cell vitality, the colometric assay CCK8 (Dojindo EU GmbH, Germany) was used and photometric absorption was recorded at 450 nm with an ELISA reader (Multiskan FC, Thermo Scientific, USA). Two additional samples (blank and copper) were used as negative and positive controls.

2.3 Hemocompatibility

2.3.1 Platelet adhesion

The adhesion of platelets on material surfaces is a key phenomenon in the formation of thrombi and a fundamental aspect of hemocompatibility. Platelets were isolated from freshly withdrawn blood through two-step centrifugation (6 min at 1750 g and 9 min at 2250 g). Citrate was used as the anticoagulant. The platelet suspension’s concentration was adjusted to approximately 500 × 103/μl and incubated for an hour with the material samples in a flow chamber. Incubation under physiological flow conditions is essential for the investigation of platelets, because their adhesion is triggered by generated shear rates [11]. After incubation, the quantification of adhered platelets on the sample surface was carried out with the acid phosphatase (ACP) assay, as described by Bellavite et al. [12]. The colorimetric substrate (p-nitrophenyl phosphate) was purchased from Sigma Aldrich Chemie GmbH, Germany, and the photometric measurement was conducted with an ELISA reader (Multiskan FC, Thermo Scientific, USA).

A SEM analysis (JSM-6390, JEOL GmbH, Germany) was carried out after incubation of the platelet suspension under the same conditions as those for the ACP assay. The samples were fixed 24 h in 3% glutaraldehyde solution and water was removed with an ascending alcohol series (50% 15 min, 70% 15 min, 80% 15 min, 99% 12 h). The samples were then coated with a 7 nm gold layer.

2.3.2 Thrombin generation

Activation of the coagulation cascade after contact with a biomaterial is another important aspect of hemocompatibility. The thrombin generation assay (TGA) was carried out in this study. Unlike the platelet adhesion test, this experiment should be conducted under static flow conditions. It has indeed been shown that the coagulation cascade can only be initiated under static or low flow conditions [13] so that thrombin production is greatest in stagnant plasma [14]. Freshly withdrawn blood with citrate as anticoagulant was processed into platelet-poor plasma through two centrifugation steps (6 min at 1750 g and 9 min at 2250 g). The TGA test was carried out as described by van Oeveren et al. [15]. The colorimetric substrate (S2238™) was purchased from Haemochrom Diagnostica GmbH, Germany, and the photometric measurement was conducted with an ELISA reader (Multiskan FC, Thermo Scientific, USA).

3 Results

3.1 Suitability as implant material

3.1.1 Long-term stable wettability

Investigation of the wetting behavior showed that all the PEG-based modifications resulted in a strong diminution of the contact angle (under 90°; unmodified silicone 119°). Moreover, hydrophilic behavior was stable during the 2-month storage period in SBF (see Figure 1), showing that the modifications suffer neither from the so-called hydrophobic recovery nor were they degraded during storage.

Figure 1: Contact angle with distilled water before and after storage of the sample in SBF (n =10).
Figure 1:

Contact angle with distilled water before and after storage of the sample in SBF (n =10).

3.1.2 Cytotoxicity

According to ISO 10993-5, a reduction of cell vitality under 70% of the value of the blank sample means that the materials exhibit a cytotoxic effect. Consequently, all tested samples could be considered noncytotoxic (see Figure 2).

Figure 2: Cytotoxicity of the samples (n =10).
Figure 2:

Cytotoxicity of the samples (n =10).

3.2 Hemocompatibility

3.2.1 Platelet adhesion

Regarding this aspect of hemocompatibility, it could be shown that all PEG-based modifications caused a significant reduction of platelet adhesion onto the surface. The number of adhered platelets was reduced by more than 70% compared to the unmodified silicone samples (see Figure 3).

Figure 3: Platelet adhesion normalized to reference sample, ACP assay (n =8). *p < 0.5, **p < 0.1, ***p < 0.05, ****p < 0.001 (Kruskal-Wallis ANOVA).
Figure 3:

Platelet adhesion normalized to reference sample, ACP assay (n =8). *p < 0.5, **p < 0.1, ***p < 0.05, ****p < 0.001 (Kruskal-Wallis ANOVA).

The SEM analysis confirmed those findings. Numerous adhered platelets in an advanced stage of adhesion (spreading of pseudopodia) were found on the unmodified silicone sample. Conversely, very few platelets were found on the PEG-modified surfaces (see Figure 4).

Figure 4: SEM images (×1500) of the pure silicone surface “Sil” (left) and the modified silicone surface “HTV+PEG” (right) after incubation with platelets. The white shapes on the surface are adhered platelets. The white bar corresponds to 10 μm.
Figure 4:

SEM images (×1500) of the pure silicone surface “Sil” (left) and the modified silicone surface “HTV+PEG” (right) after incubation with platelets. The white shapes on the surface are adhered platelets. The white bar corresponds to 10 μm.

3.2.2 Thrombin generation

Investigation of thrombin generation showed that the unmodified silicone reference did not exhibit pronounced activation of blood plasma (see Figure 5). As a consequence, it was necessary to verify that the modifications did not induce an additional activation. Only the positive control (surface coating with acid function) provoked very strong thrombin generation (12 times greater than that of the reference).

Figure 5: Thrombin generation normalized to reference sample, ACP assay (n =7). *p < 0.5, **p < 0.1, ***p < 0.05, ****p < 0.001 (Kruskal-Wallis ANOVA).
Figure 5:

Thrombin generation normalized to reference sample, ACP assay (n =7). *p < 0.5, **p < 0.1, ***p < 0.05, ****p < 0.001 (Kruskal-Wallis ANOVA).

4 Conclusion

In this study, simple PEG-based modifications of silicone elastomer that exhibit long-term stable hydrophilic properties were introduced. Two modified silicones (one with an additive compound and the other with a surface coating) are well suited for use in the blood stream, because they do not induce cytotoxic effects and improve hemocompatibility. Moreover, those materials can be industrially processed as their properties were stable after post curing and sterilization. The additive compound is particularly interesting, because it does not require an additional processing step for the HTV silicone. For example, this new material could be easily extruded to form a catheter or tubing for dialysis and extracorporeal circulation.

Author’s Statement

Research funding: The author state no funding involved. Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent has been obtained from all individuals included in this study. Ethical approval: The research related to human use complies with all the relevant national regulations, institutional policies and was performed in accordance with the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.

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Published Online: 2016-9-30
Published in Print: 2016-9-1

©2016 Cécile Boudot et al., licensee De Gruyter.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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https://doi.org/10.1515/cdbme-2016-0008
Schlagwörter für diesen Artikel
cytotoxicity; hemocompatibility; long-term stability; polyethylene glycol; Silicone elastomer; wettability
Creative Commons
BY-NC-ND 4.0

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  38. Automatic detection and mapping of double potentials in intracardiac electrograms
  39. Modeling the pelvic region for non-invasive pelvic intraoperative neuromonitoring
  40. Postprocessing algorithm for automated analysis of pelvic intraoperative neuromonitoring signals
  41. Best practice: surgeon driven application in pelvic operations
  42. Vasomotor assessment by camera-based photoplethysmography
  43. Classification of morphologic changes in photoplethysmographic waveforms
  44. Novel computation of pulse transit time from multi-channel PPG signals by wavelet transform
  45. Efficient design of FIR filter based low-pass differentiators for biomedical signal processing
  46. Nonlinear causal influences assessed by mutual compression entropy
  47. Comparative study of methods for solving the correspondence problem in EMD applications
  48. fNIRS for future use in auditory diagnostics
  49. Semi-automated detection of fractional shortening in zebrafish embryo heart videos
  50. Blood pressure measurement on the cheek
  51. Derivation of the respiratory rate from directly and indirectly measured respiratory signals using autocorrelation
  52. Left cardiac atrioventricular delay and inter-ventricular delay in cardiac resynchronization therapy responder and non-responder
  53. An automatic systolic peak detector of blood pressure waveforms using 4th order cumulants
  54. Real-time QRS detection using integrated variance for ECG gated cardiac MRI
  55. Preprocessing of unipolar signals acquired by a novel intracardiac mapping system
  56. In-vitro experiments to characterize ventricular electromechanics
  57. Continuous non-invasive monitoring of blood pressure in the operating room: a cuffless optical technology at the fingertip
  58. Application of microwave sensor technology in cardiovascular disease for plaque detection
  59. Artificial blood circulatory and special Ultrasound Doppler probes for detecting and sizing gaseous embolism
  60. Detection of microsleep events in a car driving simulation study using electrocardiographic features
  61. A method to determine the kink resistance of stents and stent delivery systems according to international standards
  62. Comparison of stented bifurcation and straight vessel 3D-simulation with a prior simulated velocity profile inlet
  63. Transient Euler-Lagrange/DEM simulation of stent thrombosis
  64. Automated control of the laser welding process of heart valve scaffolds
  65. Automation of a test bench for accessing the bendability of electrospun vascular grafts
  66. Influence of storage conditions on the release of growth factors in platelet-rich blood derivatives
  67. Cryopreservation of cells using defined serum-free cryoprotective agents
  68. New bioreactor vessel for tissue engineering of human nasal septal chondrocytes
  69. Determination of the membrane hydraulic permeability of MSCs
  70. Climate retainment in carbon dioxide incubators
  71. Multiple factors influencing OR ventilation system effectiveness
  72. Evaluation of an app-based stress protocol
  73. Medication process in Styrian hospitals
  74. Control tower to surgical theater
  75. Development of a skull phantom for the assessment of implant X-ray visibility
  76. Surgical navigation with QR codes
  77. Investigation of the pressure gradient of embolic protection devices
  78. Computer assistance in femoral derotation osteotomy: a bottom-up approach
  79. Automatic depth scanning system for 3D infrared thermography
  80. A service for monitoring the quality of intraoperative cone beam CT images
  81. Resectoscope with an easy to use twist mechanism for improved handling
  82. In vitro simulation of distribution processes following intramuscular injection
  83. Adjusting inkjet printhead parameters to deposit drugs into micro-sized reservoirs
  84. A flexible standalone system with integrated sensor feedback for multi-pad electrode FES of the hand
  85. Smart control for functional electrical stimulation with optimal pulse intensity
  86. Tactile display on the remaining hand for unilateral hand amputees
  87. Effects of sustained electrical stimulation on spasticity assessed by the pendulum test
  88. An improved tracking framework for ultrasound probe localization in image-guided radiosurgery
  89. Improvement of a subviral particle tracker by the use of a LAP-Kalman-algorithm
  90. Learning discriminative classification models for grading anal intraepithelial neoplasia
  91. Regularization of EIT reconstruction based on multi-scales wavelet transforms
  92. Assessing MRI susceptibility artefact through an indicator of image distortion
  93. EyeGuidance – a computer controlled system to guide eye movements
  94. A framework for feedback-based segmentation of 3D image stacks
  95. Doppler optical coherence tomography as a promising tool for detecting fluid in the human middle ear
  96. 3D Local in vivo Environment (LivE) imaging for single cell protein analysis of bone tissue
  97. Inside-Out access strategy using new trans-vascular catheter approach
  98. US/MRI fusion with new optical tracking and marker approach for interventional procedures inside the MRI suite
  99. Impact of different registration methods in MEG source analysis
  100. 3D segmentation of thyroid ultrasound images using active contours
  101. Designing a compact MRI motion phantom
  102. Cerebral cortex classification by conditional random fields applied to intraoperative thermal imaging
  103. Classification of indirect immunofluorescence images using thresholded local binary count features
  104. Analysis of muscle fatigue conditions using time-frequency images and GLCM features
  105. Numerical evaluation of image parameters of ETR-1
  106. Fabrication of a compliant phantom of the human aortic arch for use in Particle Image Velocimetry (PIV) experimentation
  107. Effect of the number of electrodes on the reconstructed lung shape in electrical impedance tomography
  108. Hardware dependencies of GPU-accelerated beamformer performances for microwave breast cancer detection
  109. Computer assisted assessment of progressing osteoradionecrosis of the jaw for clinical diagnosis and treatment
  110. Evaluation of reconstruction parameters of electrical impedance tomography on aorta detection during saline bolus injection
  111. Evaluation of open-source software for the lung segmentation
  112. Automatic determination of lung features of CF patients in CT scans
  113. Image analysis of self-organized multicellular patterns
  114. Effect of key parameters on synthesis of superparamagnetic nanoparticles (SPIONs)
  115. Radiopacity assessment of neurovascular implants
  116. Development of a desiccant based dielectric for monitoring humidity conditions in miniaturized hermetic implantable packages
  117. Development of an artifact-free aneurysm clip
  118. Enhancing the regeneration of bone defects by alkalizing the peri-implant zone – an in vitro approach
  119. Rapid prototyping of replica knee implants for in vitro testing
  120. Protecting ultra- and hyperhydrophilic implant surfaces in dry state from loss of wettability
  121. Advanced wettability analysis of implant surfaces
  122. Patient-specific hip prostheses designed by surgeons
  123. Plasma treatment on novel carbon fiber reinforced PEEK cages to enhance bioactivity
  124. Wear of a total intervertebral disc prosthesis
  125. Digital health and digital biomarkers – enabling value chains on health data
  126. Usability in the lifecycle of medical software development
  127. Influence of different test gases in a non-destructive 100% quality control system for medical devices
  128. Device development guided by user satisfaction survey on auricular vagus nerve stimulation
  129. Empirical assessment of the time course of innovation in biomedical engineering: first results of a comparative approach
  130. Effect of left atrial hypertrophy on P-wave morphology in a computational model
  131. Simulation of intracardiac electrograms around acute ablation lesions
  132. Parametrization of activation based cardiac electrophysiology models using bidomain model simulations
  133. Assessment of nasal resistance using computational fluid dynamics
  134. Resistance in a non-linear autoregressive model of pulmonary mechanics
  135. Inspiratory and expiratory elastance in a non-linear autoregressive model of pulmonary mechanics
  136. Determination of regional lung function in cystic fibrosis using electrical impedance tomography
  137. Development of parietal bone surrogates for parietal graft lift training
  138. Numerical simulation of mechanically stimulated bone remodelling
  139. Conversion of engineering stresses to Cauchy stresses in tensile and compression tests of thermoplastic polymers
  140. Numerical examinations of simplified spondylodesis models concerning energy absorption in magnetic resonance imaging
  141. Principle study on the signal connection at transabdominal fetal pulse oximetry
  142. Influence of Siluron® insertion on model drug distribution in the simulated vitreous body
  143. Evaluating different approaches to identify a three parameter gas exchange model
  144. Effects of fibrosis on the extracellular potential based on 3D reconstructions from histological sections of heart tissue
  145. From imaging to hemodynamics – how reconstruction kernels influence the blood flow predictions in intracranial aneurysms
  146. Flow optimised design of a novel point-of-care diagnostic device for the detection of disease specific biomarkers
  147. Improved FPGA controlled artificial vascular system for plethysmographic measurements
  148. Minimally spaced electrode positions for multi-functional chest sensors: ECG and respiratory signal estimation
  149. Automated detection of alveolar arches for nasoalveolar molding in cleft lip and palate treatment
  150. Control scheme selection in human-machine- interfaces by analysis of activity signals
  151. Event-based sampling for reducing communication load in realtime human motion analysis by wireless inertial sensor networks
  152. Automatic pairing of inertial sensors to lower limb segments – a plug-and-play approach
  153. Contactless respiratory monitoring system for magnetic resonance imaging applications using a laser range sensor
  154. Interactive monitoring system for visual respiratory biofeedback
  155. Development of a low-cost senor based aid for visually impaired people
  156. Patient assistive system for the shoulder joint
  157. A passive beating heart setup for interventional cardiology training
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Heruntergeladen am 7.9.2025 von https://www.degruyterbrill.com/document/doi/10.1515/cdbme-2016-0008/html
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