Resorbable polymers have been established for several decades in biomedical applications. The most frequently used resorbable polymers are still the aliphatic polyesters polylactides (PLA) and polyglycolid (PGA). The mechanism of polyester degradation is well known today. Polymer chains are degraded by hydrolytic scission of ester linkages in the polymer backbone by creating carboxylic end groups. These acidic carboxyl end groups decrease the pH of the implant environment [1–5]. This pH drop under the physiological value of 7.4, is one of the main concerns regarding the use of aliphatic polyesters for implants. The low pH value may cause inflammation in the host tissue [6–10]. A promising approach to realise pH optimised degradation is the incorporation of neutralising buffer systems into the polyesters. Furukawa et al. and Heidemann et al. have already investigated the incorporation of hydroxyapatite and sodium hydrogenphosphate to aliphatic polyesters. All results are based on injection or compression moulded parts respectively foils [11–25]. The incorporation of buffer systems into melt spun fibres was investigated from Schuster et al. In various works the incorporation of buffer systems in fibres could be shown [26–28]. A significant improvement of the sudden pH drop during the degradation of PLA/PGA devices, cannot be detected in none of the studies. Moreover, the required buffer amount cannot be incorporated in to the fibres via the melt spinning process.
To investigate the knowledge base in the context of absorbable polymers for medical use, we conducted a publication search in the Thomson-Reuters Web of Science Core Collection. This collection comprises most journals of relevant scientific disciplines (e.g. Medicine, Materials Science, Biotechnology, Biochemistry). A pairwise search of content-relevant keyword combinations yielded a total number of 2364 publications in this context. However, exemplary for the scope of this study, topics relevant to pH-regulation in degradable fibres yield only six publications. Considering the potential benefits of pH-optimised degradation, this publication gap signifies the need for new research in the field of biodegradable fibre materials.
PLA fibres with CaCO3, Mg(OH)2 and VCL/AAEM/Vlm (8 mol%)-microgels with an amount of 1.25 wt% could be produced successfully. The mechanical properties are shown in Table 1.
Figure 1 shows an SEM image of the as-spun fibres.
The shrunken profile of the fibre is typical for wet spun fibres and can be seen clearly. In order to obtain a buffering effect, there has to be a sufficient microgel concentration within the polymer. Results of the degradation tests of PLA foils with different amounts of microgel show, that 5 wt% microgel is needed to achieve a constant pH-level at 7.4 during the degradation of PLA (Figure 2).
XTT testing of human umbilical vein endothelial cells (HUVECs) reveals similar formazan absorption when exposed to non-dried PLA and pre-dried PLA, respectively, as compared to control. After exposure to both, non-dried and pre-dried PLA, cells display a vital cellular morphology as defined by the respective characteristic cell culture criterions including the characteristic flat, cobblestone like morphology of mature HUVECs .
Several mathematical models have been implemented to describe and analyse the pH level in the vicinity of the polymer. Among them was a Monte Carlo based model introduced by Han  and a reduced model first presented by Wang et al. . In the course of degradation, the concentration of oligomers and even monomers increases. Their diffusion into the environment and the resulting drop of the pH levels are described. The binding of the hydrogen ions in the pH buffer molecules occurs inside the microgel structures. The chemical principles and mathematical description of such sort of buffers is given in detail for poly(N-vinylimidazole) gels by Horta and Piérola . We applied these principles to our microgel and calculated the equilibrium pH-level in an aqueous solution of hydrochloric acid after adding a certain amount of the microgel. Thereby we varied the initial pH-level and compared the calculated pH-level with experimental data (Figure 3).
Microgel-functionalised fibres with pH-optimised degradation behaviour are a promising approach for a wide range of medical applications.
Within this study, a novel manufacturing process was established to produce microgel-functionalised fibres using the dry spinning process in a pilot scale. The biocompatibility of both non-dried and pre-dried PLA was evidenced (Figure 4). The mechanical properties of the functionalized fibres are close to dry and melt spun PLA fibres . The incorporated microgel leads to an enhancement of the mechanical properties, especially the elastic properties. Normally PLA fibres are stiff, brittle and exhibit reduced elasticity. The microgel is able to increase the space between PLA chains which eases the movement of polymeric chains to each other. This correlation has been described in the literature for PLA / PEG blends, too . The connection between microgel amount and elasticity of the fibre needs to be investigated. Further processing in the textile process chain must be examined in further works. Taking the achieved tensile strength into account, only the production of nonwovens and braided structures will be possible. Confirmation of the buffering effect and the maximum required dosage of the microgel necessary for a constant pH-value were obtained from degradation experiments of PLA foils. In comparison to the degradation studies from Furukawa et al. and Heidemann et al. we detect a constant pH level in our study. A pH drop is not registered in the first 75 days. Current research is focused on the optimisation of manufacturing processes to increase microgel concentration and to improve process reproducibility. Investigations of degradation behaviour especially with regard to the changes in mechanical properties of the functionalised fibres are in progress.
Furthermore within this study a mathematical model was established in order to describe and analyse the pH level in the vicinity of the polymer. This mathematical description of pH buffering with microgels is currently being integrated into a degradation model.
Materials and methods
The polymer fibres were manufactured on a piston-lab-spinning machine from Fourné Polymertechnik GmbH (Alfter-Impekoven, Germany). As matrix polymer the Polylactide (PLA) Ingeo™ Biopolymer 6201D by NatureWorks (Blair, NE, USA) is used. Chloroform/toluene in a ratio 3:1 was selected as solvent. The solubility of PLA in chloroform/toluene, to produce fibres in a dry spinning process, amounts to 26 wt%. The polymer was dissolved by stirring for 16 h at RT. The solution was spun at lab temperature with an extrusion speed of 0.75 cm3/min. Imaging techniques that are used are, on the one hand bright field microscopy, and on the other hand scanning electron microscopy (SEM). Optical bright field microscopy is carried out with the microscope DDM4000 M by Leica Microsystems CMS GmbH (Wetzler, Germany). The SEM measurements are carried out with the electron microscope Leo 1450 VP by Carl Zeiss AG (Oberkochen, Germany). The degradation experiments were performed in compliance with ISO 13781. The samples were stored in an oven from Thermo Fisher Scientific Inc. (Waltham, MA, USA), model Heraeus Function Line at 37°C for the respective period of time. The duration of the degradation test was 77 days and the designated material analysis was carried out on different days. The stress-strain behaviour was measured by using a Z2.5 from Zwick GmbH & Co. KG (Ulm, Germany) in compliance with DIN EN ISO 2062 and under standardised climate conditions according to DIN EN ISO 139. The pH-Value was determined by a Seven Easy pH S20 device from Mettler-Toledo AG (Greifensee, Schweiz).
The VCL/AAEM/Vlm (8 mol%)-microgels were synthesised via precipitation polymerisation according to the literature procedure . The polymerisation was carried out in a double wall glass reactor. VCL (1.75 g, 139.2 g·mol-1), and BIS (0.06 g, 154.2 g·mol 1) were dissolved in distilled water (150 mL). AAEM (0.293 mL, 214.22 g·mol-1) and VIm (0.112 mL, 94.11 g·mol-1) were added to the solution. The mixture was stirred at 70°C, and evacuated with nitrogen for 1 h. Subsequently, AMPA was added to the solution and the polymerization could be initiated. The reaction was performed for a period of 5 hours. After the polymerization, The VCL/AAEM/Vim (8 mol%)-microgels were purified by dialysis in water for 5 days, and afterwards dried by lyophilisation.
All measured values were normalized to the formazan absorption of vital cells incubated on Vicryl-Mesh (viability=100%). Always, n=12 samples were analysed. The fibre samples were sterilised (UV-Light 15 min) placed in 96-well cell culture plates (BD Bioscience, Franklin Lakes, NJ, USA) and were pre-wetted with PBS or with cell culture medium. After removal of the supernatant, 20,000 freshly harvested cells in 100 μL cell culture medium was seeded onto fibre samples and incubated for 48 h. For viability testing, cell culture medium was exchanged and 50 μL XTT reagent per well was added. After 4–24 h of incubation, 100 μL of the supernatant were transferred to 96-well microtiter plates (Nunc, Roskilde, Denmark) and measured at 450 nm. Cell attachment and morphology was visualised every 24 h and prior to addition of XTT (Olympus CK2, Olympus GmbH, Hamburg, Germany).
The current research project “pHaser” is funded by the Excellence Initiative of the German Federal and State Governments.
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Published Online: 2015-11-12
Published in Print: 2015-11-01