Oxygen-releasing biomaterials
Steg, Hilde
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Publication date: 2018
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Steg, H. (2018). Oxygen-releasing biomaterials. Rijksuniversiteit Groningen.
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Control of oxygen-release from
peroxides using polymers
Hilde Steg, Arina T Buizer, Willem Woudstra, Albert G Veldhuizen, Sjoerd K Bulstra, Dirk W Grijpma, Roel Kuijer
Published in Journal of Materials Science: Materials in Medicine 2015; 26 (7) 207
Abstract
An important limitation in cell therapy for the regeneration of tissue is the initial lack of oxygen. After implantation of large 3D cell-seeded structures, cells die rather than contribute to tissue regenerating. Oxygen-releasing materials were tested to improve cell survival and growth after implantation. Calcium peroxide (CaO2) in a polymer matrix was used as source of oxygen. Two polymers were tested in order to slow down and extend the period of oxygen-release, poly(D,L-lactic acid) and poly(lactic-co-glycolic acid). Compared to CaO2 particles, both releasing systems showed an initially higher and shorter oxygen-release. Human mesenchymal stromal cells cultured on casted films of these oxygen-releasing composites required catalase to proliferate, indicating the production of cytotoxic hydrogen peroxide as intermediate. Poly(D,L-lactic acid) and poly(lactic-co-glycolic acid) are less suited for slowly oxygen-releasing materials. Catalase was able to reduce the cytotoxic effect of H2O2.
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Introduction
Cell therapy using autologous cells for replacing malfunctioning tissue is hampered by the lack of vasculature at the implantation site, resulting in cell death of the implanted cells1. Important factors causing cell death are considered to be the limited amounts of oxygen and nutrients and the disability of cells to get rid of waste products. A potential solution for the lack of oxygen may be the use of composite oxygen-delivering scaffold materials, with peroxide salts as source of oxygen2. These materials should deliver oxygen to cells or tissue for a prolonged period of time such that implanted cells survive and contribute to tissue repair, but without interfering with angiogenesis, which is induced in hypoxic conditions3–7. Such composites should provide cell survival throughout the scaffold, thereby improving tissue restoration or repair. Oxygen-release should last until the angiogenic process is complete and a new functional vascular network is produced.
Peroxides provide oxygen by reaction with water (Equation 1). The reaction intermediate, hydrogen peroxide (Equation 2) is considered to be a cytotoxic agent. Mammalian cells have several defence mechanisms to help convert H2O2 into oxygen and water and can deal with low concentrations of hydrogen peroxide7.
𝐶𝑎𝑂2+ 2𝐻2𝑂 ⇌ 𝐶𝑎(𝑂𝐻)2+ 𝐻2𝑂2
[Equation 1]
2𝐻2𝑂2 ⇌ 2𝐻2𝑂 + 𝑂2(Catalase)[Equation 2]
A careful balance between oxygen delivery, angiogenesis and cytotoxicity is required. In this study oxygen-release from composite materials, consisting of CaO2 powder embedded in biodegradable poly-(lactic acid)(PLA) and poly(lactic-co-glycolic acid)(PLGA) (Figure 1), were evaluated for their oxygen-delivering capacity and their cytotoxicity to human bone marrow mesenchymal stromal cells (hMSC). The polymer matrix was intended to act as a barrier for both inflow of water and outflow of active agent, slowing down the reaction and reducing cytotoxicity. Thus, we hypothesize that PLA and PLGA polymer barriers prolong oxygen-release and reduce cytotoxicity5,8,9. Since PLGA is faster degrading than PLA, the PLGA-based composite is expected to show faster oxygen-release.
Materials and Methods
Solutions of Poly(DL-lactic-co-glycolide) (MW 153,000 g/mol)(PLGA) and Poly(DL-Lactide) (PDL20 50/50, MW 400,000 g/mol)(PLA) (PURAC, Gorinchem, the Netherlands) in chloroform were prepared at 10%(w/v). Five percent (w/w) CaO2 (75% purity, 200 mesh, <75µm; Sigma-Aldrich, Zwijndrecht, Netherlands) powder was dispersed and stirred vigorously. For oxygen measurements, 1ml of the suspension was pipetted in a 50mL vial and dried at room temperature for 48hours and in vacuum for another 24hours. Oxygen-delivery was measured at 37°C in 35mL deoxygenated Simulated Body Fluid(SBF)10 with a WTW Oxi 3310 oxygen meter (Weilheim, Germany). The oxygen measurement set-up was an open system, oxygen-free through a continuous in- and outflow of N2 gas. Oxygen-release from CaO2 alone was assessed from 5mg CaO2 packed in a porous filter paper placed at the bottom of the flask, which was then filled with 35mL SBF.
For cell culture, 15mm cover glasses were coated with 250µL suspension and dried as described above. Control materials were produced from 10% polymer solutions, not containing CaO2. hMSC, isolated from bone marrow of patients receiving a total hip replacement (Buizer et al)11, were cultured in alpha-MEM (Life technologies Europe, Bleiswijk, Netherlands) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS)(Life Technologies), 0.2mM Ascorbic-acid-2-phosphate (Sigma-Aldrich) and 1% antibiotic-antimycotic solution (Life Technologies) in an humidified atmosphere supplemented with 5% CO2. To assess cytotoxicity, passage 3 hMSC 10,000 /well were seeded in a 24-wells plate containing PLA, PLA/CaO2, PLGA, PLGA/CaO2-coated coverglasses and cultured in normoxic (21% O2) or hypoxic conditions (0.1% O2 in an InVivo2 200 incubator (Baker-Ruskinn, Bridgend, UK). Where indicated, catalase (Sigma-Aldrich) was added to the cell cultures at a concentration of 100U/ml.
Cell viability was assessed with an XTT assay, according to the instructions of the manufacturer (Applichem, GmbH, Darmstadt, Germany). The absorbance at 460nm and 690nm were read using a Fluostar optima microplate reader (BMG labtech, De Meern, the Netherlands).
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Statistical analyses were performed using a Univariate Anova and post-hoctests in SPSS 20.0.0.2. Time, material and the presence of catalase were the variables assessed.
Results and discussion
Oxygen-release of composite materials and calcium peroxide particles was determined in an open system, flushed with nitrogen at 37°C. Embedding of CaO2 in PLA and PLGA resulted in a faster release of oxygen compared to the release from CaO2 crystals alone (Figure 2).
Figure 2: Oxygen-release in time from CaO2 and CaO2 crystals embedded in polylactide polymer.
Oxygen-release measurements were performed in an ‘open’, anoxic system at 37°C.
Embedding of CaO2 crystals in polydimethylenesiloxane (PDMS) to reduce the amount of water influx and H2O2 outflow, resulted in a very effective slow-release system for oxygen5. However, PDMS is a non-degradable material and has been shown to have little cell adhesive properties5,12. Hydrolysis of the lactide-based polymers may have resulted in lowering of the pH, thereby inducing a higher solubility of the intermediate Ca(OH)2, shifting the reaction towards H2O2 production13. 0 1 2 3 4 5 6 0 1 2 3 4 Ox yg en ( m g/ L) Time (days) PLGA CaO₂ P(DL)LA CaO₂ CaO₂
PLGA-based composite materials for oxygen-release were previously used by Harrison4 and Oh3, either combined with Na2CO3·1.5H2O2 or with CaO2. The observed oxygen-release kinetics of these studies are difficult to compare to our data, since these investigators used a closed system to assess oxygen-release and our setup was an open system more resembling an in vivo situation3.
Furthermore, the production method of the oxygen-delivering composite differs, which may have decreased the influx of water in their material as compared to ours. Our setup, an open system, did not allow for assessment of the total amount of oxygen delivered by the material.
A B
Figure 3: Histogram representing absorbance values of the XTT viability assay. Human mesenchymal stromal cells were seeded onto oxygen-releasing and non-oxygen-releasing materials and cultured in a hypoxic (0.1 %O2) environment. To some cultures catalase was added to the culture medium
to reduce the H2O2 concentration, thereby decreasing cytotoxicity. Cells were cultured for 1, 4, or
7 days. Statistical significant differences are shown using *. Furthermore, differences between with and without catalase are statistical significant for PLA/CaO2 and for PLGA/CaO2 at t=4 and t=7.
Without catalase the difference between oxygen-releasing and non-oxygen-releasing materials is overall statistically significant. With catalase the differences between PLGA and PLGA/CaO2 are
significant, PLA vs. PLA/CaO2 is not significant except for t=7.
hMSC grown on PLGA or PLA surfaces showed a better viability than hMSC grown on CaO2-composite films (Figure 3). hMSC grown on PLA/CaO2 constructs exhibited XTT values of 0.508 (t=1) and 0.877 (t=7), whereas the XTT conversion by hMSC on PLGA/CaO2 constructs revealed values of 0.244 (t=1) and 0.91 (t=7). If the conversion of XTT correlates with the number of
0 1 2 3 PLA PLA CaO₂ PLGA PLGA CaO₂ TCPS absorba nce ( 46 0 nm ) PLA PLA CaO₂ PLGA PLGA CaO₂ TCPS with catalase t=1 t=4 t=7 * [ * [ * [ * [ * [ * [ * [ * [ * [
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cells, then cells grew faster on PLGA/CaO2 then on PLA/CaO2. Initial adhesion ofhMSC appeared to be better on PLA/CaO2. The burst release of oxygen from especially PLGA/CaO2 might have caused this decrease in cell viability at t=1. The cells that survived the relatively high H2O2 concentrations then grow better in hypoxic conditions on PLGA/CaO2 surfaces then on PLA/CaO2 surfaces. Catalase significantly improved the ability of hMSC to convert XTT; the XTT values for cells cultured on PLGA/CaO2 without catalase varied between 0.127 and 0.253, with catalase the values were 0.24 and 0.91 (P<0.05).
hMSC were not much affected by the hypoxic conditions chosen, whether grown on polymer constructs or on tissue culture polystyrene (TCPS), the conversion of XTT appeared even to have increased after 7 days. (One should keep in mind that the reduction of conversion of XTT is catalysed by dehydrogenases and reductases in mitochondria, which increase in activity when cell metabolism changes from aerobic to anaerobic14,15). At longer culture periods (>= 10 days) at 0.1% oxygen hMSC on TCPS and polymer surfaces (no CaO2) did show a reduced viability, illustrated by decreasing cell numbers (data not shown). A similar resistance of MSC (from sheep) to hypoxia was observed by Deschepper et al. when sMSC were cultured in medium supplemented with extra glucose16. These investigators varied glucose concentrations in the culture medium but did not refresh the medium during the hypoxic (1%) culturing period of 12 days. Nevertheless, sMSC did hardly show signs of cell death. Similar effects were observed in our study using normal concentrations of glucose but refreshing the medium twice a week; the lack of glucose caused by culturing in a closed system was probably not reached.
The data obtained in this study did not support our hypothesis. Incorporation of CaO2 in PLA or PLGA did not result in a slow release system for oxygen. The most probable cause is the acidity of the degradation products of the polymer. The intermediate reaction product Ca(OH)2 may have stimulated polymer hydrolysis by increasing the pH17,18. Both PLA and PLGA appear to have a limited suitability for use in a slow oxygen-release system.
Conclusions
Biodegradable, oxygen-releasing composite materials prepared by embedding CaO2 particles embedded in either PLA or PLGA polymers show enhanced oxygen-release compared to free CaO2. The polymer-peroxide constructs appeared to be moderately cytotoxic. Catalase was able to reduce cytotoxicity, which is indicative for the role of H2O2 in this process. PLA and PLGA polymers are less suited for slow-releasing oxygen systems.
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