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Environmental fate and effect of biodegradable electro-spun scaffolds (biomaterial)-a case study

Environmental fate and effect of biodegradable electro-spun scaffolds (biomaterial)-a case study Poly-ε-caprolactone (PCL) based medical devices are increasingly produced and thus, their presence in the environment is likely to increase. The present study analysed the biodegradation of PCL electro-spun scaffolds (alone) and PCL electro- spun scaffolds coated with human recombinant (hR) collagen and Bovine Achilles tendon (BAT) collagen in sewage sludge and in soil. Additionally, an eco-toxicological test with the model organism Enchytraeus crypticus was performed to assess environmental hazard of the produced materials in soils. The electro-spun scaffolds were exposed to activated sludge and three different soils for various time periods (0-7-14-21-28-56-180 days); subsequently the degradation was determined by weight loss and microscopical analysis. Although no toxicity occurred in terms of Enchytraeus crypticus reproduction, our data indicate that biodegradation was dependent on the coating of the material and exposure condition. Further, only partial PCL decomposition was possible in sewage treatment plants. Collectively, these data indicate that electro-spun PCL scaffolds are transferred to amended soils. 1 Introduction ought to be studied, as most wastes, they can become concentrated in sewage sludge due to discharges to urban A range of novel scaffold fabrication technologies are under wastewaters and domestic or industrial sources. New leg- intense development, including, for example, the develop- islations also impose life cycle impact assessment and ment of electro-spun meshes to be used in medical surgery development of products that would comply with low car- [1, 2]. Among the various available polymers, poly-ε- bon economy directives. caprolactone (PCL) and collagen are favoured as raw PCL (an aliphatic polyester) is generally biodegradable materials for scaffold fabrication. Indeed, numerous pro- by hydrolysis. Previous studies have shown that both the ducts based on these materials are currently clinically block length and the structure of the material are crucial for available as drug delivery systems, sutures and adhesion its biodegradation [3]. For example, previous studies have barriers, to mention only a few. Due to their increasing shown that the erosion of 2,2’-bis(2-oxazoline)-linked poly- production and use, their environmental fate and effects ε-caprolactone films increased in parallel with decreasing the PCL block length [4]. Further, degradation of PCL film by Pseudozyma japonica-Y7-09 reached maximum (93.33%) at day 15, whilst degradation of PCL foam by * J. J. Scott-Fordsmand jsf@bios.au.dk Pseudozyma japonica-Y7-09 reached 43.2% at day 30 [5]. In preclinical models, it has been shown that poly(hydro- Department of Bioscience, Aarhus University, Vejlsoevej 25, DK- xymethylglycolide-co-ε-caprolactone 3D-scaffolds lost 8600 Silkeborg, Denmark more than 60% of their weight and their molecular weight Department of Biology and CESAM, University of Aveiro, 3810- was decreased from 46.9 to 23.2 kDa within 3 months of 193 Aveiro, Portugal implantation, whilst PCL scaffolds showed no weight and Regenerative, Modular and Developmental Engineering no molecular weight loss during the same period [6]. It has Laboratory (REMODEL), Biomedical Sciences Building, National also been shown that PCL capsules with 66 kDa molecular University of Ireland Galway (NUI Galway), Galway, Ireland weight remained intact in shape for 24 months post- Science Foundation Ireland (SFI) Centre for Research in Medical implantation and they broke into low molecular weight Devices (CÚRAM), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland (8 kDa) pieces after 30 months implantation; further, 1234567890();,: 1234567890();,: 51 Page 2 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:51 tritium-labelled PCL (3 kDa molecular weight) was detected dimethy- laminopropyl]carbodiimide (EDC): N-hydroxy- in plasma 15 days after implantation and 92% of the sulfosuccinimide (NHS) in 0.05 M 2-(N-morpholino) etha- implanted radioactive tracer was detected in faeces and nesulfonic acid (MES) buffer at pH 5.5 in a 3:1:5 ratio urine 135 days post-implantation, indicating that the mate- respectively, as has been described previously [22]. The rial did not cumulate in body tissue, but completely excreted following day, three phosphate buffered saline (PBS) into the environment [7]. It is worth noting that PCL washes were carried out and the meshes were allowed to dry decomposes in sludge with a half-life of approximately 120- at room temperature. Morphological assessment was per- 150 days [8]. Therefore, the residues of the PCL will still formed using a Hitachi S-94700 scanning electron micro- remain in the sludge after its processing in the sewage scope (Hitachi High-Technologies Europe GmbH, treatment plant, and hence enter the soil compartment Germany) after gold sputter coating was applied. Materials through the disposal of the sludge in agricultural soils. Once will hereafter be named as PCL, hR and BAT respectively in the soil, bacteria and fungi will further degrade PCL [8– for simplicity. 12]. Given that over 500 papers have been published in the last 5 years using electro-spun PCL scaffolds (Source: 2.2 Degradation test in activated sludge PubMed, Terms searched: ‘PCL’ and ‘Electrospun’ at Title / Abstract), it is essential to assess the environmental degra- The test was performed according the standard guidelines dation of this new PCL architectural conformation. Degra- [23]. The activated sludge was collected from a sewage dation of collagen (used in the present study) in nature is treatment plant in Silkeborg (Denmark) at two instances also dependent on environmental conditions, which is well (April and August), and transported to the laboratory with known in forensic and archaeological science studying an adequate ventilation and temperature (20 °C ± 1). Once remains of humans and animals [13]. There are no studies in the laboratory, the sludge was sieved with a 2 mm mesh on the environmental fate of recombinant collagen. Herein, to calculate the total suspended solid concentration, which we aimed to determine the degradation of non-coated and was adjusted to 4000 mg/l with distilled water. An abiotic collagen-coated PCL electro-spun scaffolds in the envir- replicate of sludge was prepared for each sampling time by onment. Testing included the impact of sewage sludge and autoclaving it for 90 min at 121 °C and 15 psi. After cool- comparison of soils with different physico-chemical char- ing, pH of the abiotic was measured and adjusted to equal acteristics. To assess the potential biological impact of the that of the biologically active sludge. Biologically active produced scaffolds to soil invertebrates, the standard model and abiotic sludge (150 ml) were placed in 500 ml flasks, Enchytraeus crypticus [14–16] was used. and 6 pieces of 15 mg of test mesh were immersed indivi- dually inside a nylon mesh in each test vessel. The vessels were maintained with continuous shakes and a constant 2 Material and Methods temperature of 25 °C during the test. The meshes were removed after 3, 6, 11, 14, 21 and 24 days of exposure, 2.1 Test materials gently washed with distilled water and weighted (details ahead). Typical protocols for electro-spinning were utilised, as described previously [17–21]. In short, 200 mg/ml of PCL 2.3 Degradation test in soil (PC-12, Corbion, The Netherlands) were dissolved in 2,2,2- trifluoroethanol (TFE; 99.8%, Acros Organics, Ireland) and The degradation in soil was performed with three different was extruded at 100 µl/min from three syringes simulta- standard natural soils (Table 1), purchased from LUFA- neously through an 18 G stainless steel blunt needle (EFD Speyer (Germany). Test soils were dried for 24 h at 90 °C Nordson, Ireland). Upon application of high voltage and their moisture was adjusted to the 50% of the water (20 kV) between the needle and the collector (20 cm dis- holding capacity with a 1:1 mixture of deionized water and tance), the fibres were collected on a rotating mandrel (60 microbial substrate containing the native microbial and RPM). Three meshes were stacked together and compressed nematode community of the soils. The microbial substrate at Proxy Biomedical (Ireland) under optimal conditions of was based on 1 kg of test soil mixed with 2 l of deionized temperature / pressure. The compressed meshes were then water for 3 h, sieved through a 50 µm mesh and diluted 10 coated with 5 mg/ml human recombinant collagen isolated times [24, 25]. One vessel per soil type was prepared with form transgenic tobacco plants (hR Col; CollPlant®, Israel) 100 g of dried soil. Four pieces of ca. 10 mg of the test or bovine Achilles tendon collagen (BAT; Vornia Bioma- material (i.e., 1,000 mg mesh per kg dry weight) were then terials, Ireland). After coating, the meshes were allowed to placed in the soil individually inside a nylon mesh, and one air dry in a laminar flow hood at room temperature over- piece was removed after each respective sampling date: 7, night. The meshes were then cross-linked with 1-ethyl-3-[3- 14, 21 and 28 days of exposure. Journal of Materials Science: Materials in Medicine (2018) 29:51 Page 3 of 10 51 Table 1 Physico-chemical characteristics of LUFA 2.1, LUFA 2.2 and LUFA 2.4 soils including organic carbon content, pH and nitrogen content (average ± SD) and the soil type Soil parameter LUFA 2.1 LUFA 2.2 LUFA 2.4 Organic carbon (%) 0.71 ± 0.07 1.59 ± 0.13 2.03 ± 0.23 pH (0.01 M CaCl ) 4.9 ± 0.3 5.4 ± 0.2 7.3 ± 0.1 Nitrogen (%) 0.06 ± 0.01 0.17 ± 0.01 0.22 ± 0.02 Soil type Salty sand (uS) Loamy sand (lS) Clayey loam (tL) After this first experiment, a second was conducted to Afterwards, the remaining material was weighted in a evaluate the degradation of the electro-spun scaffolds in precision microbalance (0.001 ± 0.003 mg, Sartorius Micro soils amended with sludge. Sludge amendment was per- MC5-SC2). formed following the protocol of [26]. Briefly, 10 mg of To identify changes in surface area as the decomposition sludge was added to 100 g of dry soil, and after homo- progressed, it was attempted to use dye-adsorption/deso- genising, the soil moisture was adjusted to 50% of the rption techniques. However, since no consistent results were maximum water holding capacity (WHC) with deionized obtained for all the test materials, the method was dis- water. Parallel to this, a control (non-amended soil) was regarded. Scanning Electron Microscopy (SEM) was con- prepared by simply adjusting the moisture to 50% of the sidered, mainly providing qualitative structural indices, but WHC. Three pieces of 10 mg of the test material (i.e., not deemed to provide additional required quantitative 1000 mg mesh per kg dry weight) were then placed in the information, since the dry weight measures provided the soil individually, and one piece was removed at a time after required information i.e., disappearance of the PCL. 28, 56 and 180 days of exposure. 2.6 Data analysis 2.4 Toxicity test in soil One-way analysis of variance (ANOVA) followed by The test was performed according to the standard guidelines Dunnettʼs comparison post-hoc test (p ≤ 0.05) was used to of the Enchytraeid Reproduction test [14], using LUFA assess differences between control and treatments (Sigma- 2.2 soil and the test species Enchytraeus crypticus. Soil was Plot 11.0). Degradation Time (DTx) calculations were moist with deionized water adjusting the humidity to 50% performed modelling data to logistic sigmoid 2 parameters of the water holding capacity, and 25 g of moist soil was regression models, as indicated in Table 2, using the introduced in plastic vessels. The scaffolds were inserted in Toxicity Relationship Analysis Program (TRAP v1.22) each container, as explained above, in a concentration of software. 1000 mg per kg dry soil. Additionally, 6 control vessels were prepared containing 1000 mg/kg filter paper as control (VWR, filter paper No. 413). Organisms of synchronised 3 Results age (17-19 days) (for details see [15]) were transferred to each test vessel; 6 replicates were done. The exposure ran 3.1 Electro-spun scaffold characterisation for 28 days and was performed under constant temperature (20 °C) and a light regime of 16 h light/8 h dark. Water loss Microscopy analysis revealed no apparent differences and food (finely ground and autoclaved oat rolls) was between the groups in macro and micro level (Fig. 1). replenished weekly. Test endpoints include survival and Further, no significant difference (p > 0.05) in fibre diameter reproduction (number of juveniles). was observed between the groups (electro-spun alone: 2.2 ± 1.3 μm; coated with BAT Col I: 1.7 ± 0.8 μm; coated with 2.5 Determination of weight loss hR Col I: 2.1 ± 1.0 μm; n = 100 measurements). To determine weight in the above test without interference 3.2 Activated sludge exposure test from biofilm or soil particles attached to the surface of the test materials, a sonication method was used [27–29]. The The degradation rates were clearly much lower in the test materials were sonicated in deionized water for 2 min. abiotic conditions (Fig. 2). For the April sludge exposure, (Branson 5510, 50-60 Hz and 110 VAC, Fisher Scientific, abiotic conditions caused degradation of only 17% for PCL, US)todetachpossible biofilm from the electro-spun 4% for BAT and 16% for hR (21 days, 24 days was lost). scaffold, washed again and dried at 37 °C for 24 h. The exposure in biotic conditions caused a significant 51 Page 4 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:51 Table 2 Degradation time (days) for the materials tested in sludge collected in April (one value for the August test sludge for PCL hR), indicating the DT50 and DT90 (time to degrade 50% and 90%) estimates and respective model and parameters (Sigmoid 2 parameters; S: Slope; Y0: intercept) DT50 (days) DT90 (days) Model and parameters Biotic PCL 22 [18<CI<26] 33 Sigm. 2 param. S0=0,032, Y0=100; PCL (August) 116 [15<CI<896] – Sigm. 2 param. LogX S0=0,370; Y0=100 PCL hR 25 [21<CI<29] 37 Sigm. 2 param. S0=0,028; Y0=100; PCL hR (August) 125 [127<CI<376] 256 Sigm. 2 param. S0=0,0032; Y0=100; PCL BAT 20 [17<CI<23] 37 Sigm. 2 param. S0=0,035; Y0=100; Abiotic PCL n.e. n.e. – PCL hR 23 [20<CI<26] 29 Sigm. 2 param. S0=0,092; Y0=100; PCL BAT n.e. n.e. – CI 95% confidence interval Fig. 1 Stereo and scanning electron microscopy analysis revealed no apparent differences between the groups (PCL electro-spun scaffold alone (PCL) and PCL electro-spun scaffolds coated with BAT Col I (BAT) and rH Col I (hR)) Journal of Materials Science: Materials in Medicine (2018) 29:51 Page 5 of 10 51 B: August Fig. 2 Degradation represented A: April as weight loss (% initial weight) 100 100 of PCL electro-spun scaffold (PCL), PCL BAT electro-spun scaffold (BAT) and PCL hR 60 60 electro-spun scaffold (hR). a exposure from 0 to 24 days to activated sewage sludge (solid lines) and the abiotic sludge (dashed lines) collected in April. 0 0 b exposure from 0 to 180 days to 1 28 56 180 036 1114 2124 activated sewage sludge (solid time (days) time (days) lines) and the abiotic sludge (dashed lines) collected in PCL Bio hR Bio BAT Bio August PCL Ab hR Ab BAT Ab Fig. 3 Pictures of the various PCL electro-spun scaffolds at day 0 (up) and at day 24 (down) of exposure to the biotic sludge i.e., PCL, BAT and hR. Corner scale bar: 1 mm (P < 0.05) higher degradation of all materials during the 3.3 Degradation test in soil 24 days (Fig. 2): with weight loss around 50% i.e., measured 56% for BAT, 51% for PCL (51%) and 45% for hR. The The PCL electro-spun scaffolds degradation was generally time for degradation estimates can be depicted in Table 2. low, although hR showed a 20% weight loss after exposure The microscopic observations also illustrate a higher to LUFA 2.4 soil for 28 days (Fig. 4). In LUFA 2.1 and degradation on the PCL electro-spun scaffold (Fig. 3). LUFA 2.2 soils the degradation was below 6%. Fungal hyfa Regarding the long-term degradation of the electro-spun proliferation was observed in non-coated and coated PCL scaffolds tested in the sludge collected in August, it was electro-spun scaffolds in the three tested soils after 7 days of slower than in the experiment performed in April for all exposure to all the soils (Fig. 5). materials (Fig. 2). The weight loss of PCL electro-spun Longer exposures of PCL electro-spun scaffolds showed scaffold was similar for both sludge types, and concerning degradation on the first 28 days in the three soil treatments hR electro-spun scaffold, the degradation was 30% lower (microbial substrate, sludge-amended soil and soil). Sludge after 180 days in August sludge than after 28 days in April amendment caused an increased degradation of the PCL and sludge. hR compared to the soil in LUFA 2.1 soil, although BAT % start weight 51 Page 6 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:51 soil soil + microbial substrate soil + sludge amended Fig. 4 Degradation represented 100 100 100 as weight loss (% start weight) 90 90 of electro-spun scaffolds PCL, 80 80 80 hR, and BAT, in three soil types: LUFA 2.1, LUFA 2.2 and 70 70 70 LUFA 2.4. Left column: 60 60 LUFA 2.1 LUFA 2.1 LUFA 2.1 Exposure over 28 days to soils 07 14 21 28 028 56 180 028 56 180 with added microbial substrate. time (days) time (days) time (days) Middle column: Exposure over 180 days to soils (no soil + microbial substrate soil soil + sludge amended amendments). Right column: 100 100 Exposure over 180 days to soil 90 90 90 amended with August sewage 80 80 80 sludge. The regression lines to 70 70 70 the data are given per material 60 60 LUFA 2.2 60 LUFA 2.2 and soil in the table below, LUFA 2.2 showing the corresponding R 07 14 21 28 028 56 180 0 28 56 180 time (days) time (days) time (days) soil + sludge amended soil + microbial substrate soil 100 100 100 PCL 90 90 90 hR 80 80 BAT 70 70 70 60 60 60 LUFA 2.4 LUFA 2.4 LUFA 2.4 0 7 14 21 28 028 56 180 028 56 180 time (days) time (days) time (days) Soil Type BM soil + microbial substrate soil soil + sludge amended PCL -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 LUFA 2.1 hR -0,0138xdays+ 96,875, R² = 0,0042 -0,0138xdays+ 96,875, R² = 0,0042 -0,0138xdays+ 96,875, R² = 0,0042 BAT -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 PCL -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 LUFA 2.2 hR -0,0138xdays+ 96,875, R² =0,0042 -0,0138xdays+ 96,875, R² = 0,0042 -0,0138xdays+ 96,875, R² = 0,0042 BAT -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 PCL -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 LUFA 2.4 hR -0,0138xdays+ 96,875, R² =0,0042 -0,0138xdays+ 96,875, R² =0,0042 -0,0138xdays+ 96,875, R² = 0,0042 BAT -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 Fig. 5 Pictures of the PCL electro-spun (left column) (PCL), PCL electro-spun hR (central column) (hR), and PCL electro-spun BAT (left column) (BAT) after 28 days of exposure to LUFA 2.4 soil. Scale bar: 1 mm showed a similar degradation. Sludge amendment in LUFA 3.4 Toxicity test in soil 2.2 soil caused an increase in degradation only for BAT, while in LUFA 2.4, the degradation increased 20% for PCL No significant effects (p > 0.05) occurred for E. crypticus in and BAT (Fig. 4). terms of reproduction (Fig. 6). The degradation did not % start weight % start weight % start weight Journal of Materials Science: Materials in Medicine (2018) 29:51 Page 7 of 10 51 2.0 y=-0.0237x + 0.9732, R =0.0039 80 120 1.5 1.0 40 60 40 0.5 juveniles mesh 20 0.0 0 0 0 1234 0 PCL hR BAT weight loss (% initial weight) Test material Fig. 6 Results from the Enchytraeid Reproduction Test for Enchy- following this exposure (left numbers), and the correlation (or lack traeus crypticus exposed for 28 days in LUFA 2.2 soil to the PCL thereof) between the weight loss and the number of individual per electro-spun scaffold materials: PCL, hR and BAT. A: reproduction. adult B: the remaining weight (given as % of initial weight) of the mesh exceed the 2.2% of the initial weight during the 28 days of certain and unknown complexity in the process and that this the test (Fig. 6, left), and the number of juveniles per adult is not fully controlled or predictable. was not related to the degradation of the test materials (Fig. For the electro-spun scaffolds, the biodegradation during 6, right). the first 7 days did not differ between the non-coated and collagen-coated materials, but their degradation was slow probably due to the lower surface area (total or available to 4 Discussion microorganisms) in contact with the sludge, as the biotic degradation is known to occur at the surface [32]. As The present study showed the effect of the structure and the expected, the abiotic degradation was lower, and this has presence/absence and coating type of PCL materials on their been previously reported for sludge exposures [33], show- biodegradability and environmental fate. ing that decomposition was biological mediated. Degradation of PCL polymers by microorganisms in vitro The physico-chemical properties of soils are among the and in different environmental compartments has been well factors determining its microbial and fungal composition, studied, but the information of PCL is still very limited, which are the main decomposers of PCL. The differences particularly in regard to the differences between the electro- observed in biodegradation capacity related to soil type spun scaffold adaptations. Although biodegradation can be were expected although the exact combination of factors is defined by different parameter measurements such as loss of far less understood. For instance, pH is a decisive factor for mechanical properties (tensile strength, crystallization etc.), microbial enzymatic activity, since the enzymes require an CO production, morphological changes evaluated by SEM optimal pH for their correct functioning. Moreover, toler- or optical microscopy, etc [30]., the focus here was on ance to high or low pH is species/genus specific and, thus, physical disappearance, i.e., weight loss (dry weight), microbial diversity is strongly determined by pH, e.g., pH combined with optical microscopic observations to detect values below 5 decrease microbial decomposition and breakage or other types of changes in the surface. activity in soils [34, 35]. On the other hand, OC and N The current prospect is that the first biodegradation content represent growth-limiting resources and shape process occurs in sewage treatment plants, where industrial microbial community composition and activity [36, 37]. and domestic PCL discharges can be concentrated. As Accordingly, the decreasing gradient of pH, OC and N quantified here, the degradation of PCL materials is sludge- content of LUFA 2.4, 2.2 and 2.1, respectively, coincides dependent. In the present, the sludge was collected from the with the highest degradation measured in LUFA 2.4 soil, exact same sewage treatment plant at two time points, April followed by LUFA 2.2 and, finally, LUFA 2.1 soil. The and August, hence differences must be due to the varying presence of collagen coating in the PCL had an inhibiting waste water characteristics. This is not surprising and has effect on the degradability of the PCL mesh. This may be been previously observed regarding nonylphenol and because microbial collagenases are only produced after organic compound degradation [31]. In our study, the April exposure as a response, hence increasing the time needed sludge showed higher degradation efficiency than the for its degradation. On the other hand, although different August sludge. Additionally, the degradation rate was dif- bacteria genus commonly found in soils, such as Pseudo- ferent and the material-dependent degradation was also monas and Aeromonas, produce collagenase enzymes, it is distinct, e.g., the most degraded PCL material in April was not so common for soil fungi, and mainly nematophagous the least degraded in August. This indicates that there is a fungi species show collagenase activity. However, PCL No. juveniles (per adult, AV±SE) mesh weight (%) No. juveniles (per adult, AV±SE) 51 Page 8 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:51 degradation by fungi is well known. The fungal coloniza- issues, such as the ones recommended for the testing of tion of the PCL materials observed during the test indicates nano-materials, e.g., the need for longer test duration (or that biodegradation was caused by fungi to some extent, and surrogates), are obviously in the same line for biomaterials. since fungal collagenase activity was not likely to occur in soil fungi, the collagen coating could prevent fungi from degrading the PCL. The observed weight loss of electro- 5 Conclusions spun scaffolds after 28 days of exposure is similar to the degradation of disc-shaped PCL pieces in soil after 35 days This is the first time that such biomaterials have been tested [38], i.e., 5% in a soil with 80% organic matter content. for environmental hazard. Biodegradation of the tested PCL These authors also reported the importance of the PCL materials was coating- and exposure-dependent. There was structure for the degradation, with the amorphous regions a clear faster biotic than abiotic degradation. The coated being the first ones to be degraded. electro-spun materials degraded as fast (or faster) then the Overall, the degradation of PCL materials in soil was non-coated PCL electro-spun. The time required for total much slower than in the activated sludge, rendering weight degradation exceeds the time period in sewage treatment loss values below 30% after 6 months of exposure in the plants, hence partial decomposition of PCL is expected best case, having a considerably long persistency (life time) when transfer takes place to amend soils. Soil types will in soil. In addition, the biodegradation is soil-type depen- determine differences between degradation efficiency. dent, so the persistence of PCL in soil can vary, depending Over-all the degradation patterns in the soil were similar, on the physico-chemical characteristics and microbial also for hr and BAT, except for the LUFA 2.4 soils. We community present in soils. For example, the presence of recommend the range of standard natural LUFA soils as a collagen-degrading microorganisms in soils is not ubiqui- good model basis to assess these variations so far. There are tous, and only 15% of organic horizons of spruce growth technical issues, like the test material form (mesh) or the test soil samples and 37% of garden soil samples showed duration, that deserve further consideration and optimiza- microbial collagenase activity [39]. In fact, according to tion for an adequate environmental hazard assessment of present results, the enrichment of soils with microorgan- biomaterials. isms from activated sewage sludge could increase the Acknowledgements This study was supported by funds from the degradation of the PCL electro-spun scaffolds in soils European Commission in the FP7 NMP project Green nano mesh (G. decreasing its persistency in soil, as in agreement with A. No. 263289). Further support was provided by BIORIMA H2020- previous studies which showed the beneficial effect of NMBP-2017 (GA No. 760928), by CESAM (UID/AMB/50017-POCI- sludge amendment in the microbial activity of soils [40–42] 01-0145-FEDER-007638) via FCT/MCTES through national funds (PIDDAC) and the co-funding by the FEDER, within the PT2020 and the consequent increased degradation efficiency. The Partnership Agreement and Compete 2020), and by Science Founda- presence of the PCL in soil did not affect E. crypticus,an tion Ireland, Career Development Award Programme (grant agreement invertebrate soil model representative, in terms of repro- number: 15/CDA/3629); Science Foundation Ireland and the European duction. Moreover, the degradation of the test PCL and the Regional Development Fund (grant agreement number: 13/RC/2073); EU H2020, ITN award, Tendon Therapy Train Project (grant agree- juvenile production were not correlated. We cannot exclude ment number: 676338). that this is due to the low degradation level within the test period and the test period itself, which is short compared to Compliance with ethical standards the material lifetime. The conclusion is similar to studies made with microbial substrates [43–47] where PCL was Conflict of interest The authors declare that they have no conflict of not toxic. interest. 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Environmental fate and effect of biodegradable electro-spun scaffolds (biomaterial)-a case study

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Springer Journals
Copyright
Copyright © 2018 by Springer Science+Business Media, LLC, part of Springer Nature
Subject
Materials Science; Biomaterials; Biomedical Engineering; Regenerative Medicine/Tissue Engineering; Polymer Sciences; Ceramics, Glass, Composites, Natural Materials; Surfaces and Interfaces, Thin Films
ISSN
0957-4530
eISSN
1573-4838
DOI
10.1007/s10856-018-6063-3
pmid
29713809
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Abstract

Poly-ε-caprolactone (PCL) based medical devices are increasingly produced and thus, their presence in the environment is likely to increase. The present study analysed the biodegradation of PCL electro-spun scaffolds (alone) and PCL electro- spun scaffolds coated with human recombinant (hR) collagen and Bovine Achilles tendon (BAT) collagen in sewage sludge and in soil. Additionally, an eco-toxicological test with the model organism Enchytraeus crypticus was performed to assess environmental hazard of the produced materials in soils. The electro-spun scaffolds were exposed to activated sludge and three different soils for various time periods (0-7-14-21-28-56-180 days); subsequently the degradation was determined by weight loss and microscopical analysis. Although no toxicity occurred in terms of Enchytraeus crypticus reproduction, our data indicate that biodegradation was dependent on the coating of the material and exposure condition. Further, only partial PCL decomposition was possible in sewage treatment plants. Collectively, these data indicate that electro-spun PCL scaffolds are transferred to amended soils. 1 Introduction ought to be studied, as most wastes, they can become concentrated in sewage sludge due to discharges to urban A range of novel scaffold fabrication technologies are under wastewaters and domestic or industrial sources. New leg- intense development, including, for example, the develop- islations also impose life cycle impact assessment and ment of electro-spun meshes to be used in medical surgery development of products that would comply with low car- [1, 2]. Among the various available polymers, poly-ε- bon economy directives. caprolactone (PCL) and collagen are favoured as raw PCL (an aliphatic polyester) is generally biodegradable materials for scaffold fabrication. Indeed, numerous pro- by hydrolysis. Previous studies have shown that both the ducts based on these materials are currently clinically block length and the structure of the material are crucial for available as drug delivery systems, sutures and adhesion its biodegradation [3]. For example, previous studies have barriers, to mention only a few. Due to their increasing shown that the erosion of 2,2’-bis(2-oxazoline)-linked poly- production and use, their environmental fate and effects ε-caprolactone films increased in parallel with decreasing the PCL block length [4]. Further, degradation of PCL film by Pseudozyma japonica-Y7-09 reached maximum (93.33%) at day 15, whilst degradation of PCL foam by * J. J. Scott-Fordsmand jsf@bios.au.dk Pseudozyma japonica-Y7-09 reached 43.2% at day 30 [5]. In preclinical models, it has been shown that poly(hydro- Department of Bioscience, Aarhus University, Vejlsoevej 25, DK- xymethylglycolide-co-ε-caprolactone 3D-scaffolds lost 8600 Silkeborg, Denmark more than 60% of their weight and their molecular weight Department of Biology and CESAM, University of Aveiro, 3810- was decreased from 46.9 to 23.2 kDa within 3 months of 193 Aveiro, Portugal implantation, whilst PCL scaffolds showed no weight and Regenerative, Modular and Developmental Engineering no molecular weight loss during the same period [6]. It has Laboratory (REMODEL), Biomedical Sciences Building, National also been shown that PCL capsules with 66 kDa molecular University of Ireland Galway (NUI Galway), Galway, Ireland weight remained intact in shape for 24 months post- Science Foundation Ireland (SFI) Centre for Research in Medical implantation and they broke into low molecular weight Devices (CÚRAM), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland (8 kDa) pieces after 30 months implantation; further, 1234567890();,: 1234567890();,: 51 Page 2 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:51 tritium-labelled PCL (3 kDa molecular weight) was detected dimethy- laminopropyl]carbodiimide (EDC): N-hydroxy- in plasma 15 days after implantation and 92% of the sulfosuccinimide (NHS) in 0.05 M 2-(N-morpholino) etha- implanted radioactive tracer was detected in faeces and nesulfonic acid (MES) buffer at pH 5.5 in a 3:1:5 ratio urine 135 days post-implantation, indicating that the mate- respectively, as has been described previously [22]. The rial did not cumulate in body tissue, but completely excreted following day, three phosphate buffered saline (PBS) into the environment [7]. It is worth noting that PCL washes were carried out and the meshes were allowed to dry decomposes in sludge with a half-life of approximately 120- at room temperature. Morphological assessment was per- 150 days [8]. Therefore, the residues of the PCL will still formed using a Hitachi S-94700 scanning electron micro- remain in the sludge after its processing in the sewage scope (Hitachi High-Technologies Europe GmbH, treatment plant, and hence enter the soil compartment Germany) after gold sputter coating was applied. Materials through the disposal of the sludge in agricultural soils. Once will hereafter be named as PCL, hR and BAT respectively in the soil, bacteria and fungi will further degrade PCL [8– for simplicity. 12]. Given that over 500 papers have been published in the last 5 years using electro-spun PCL scaffolds (Source: 2.2 Degradation test in activated sludge PubMed, Terms searched: ‘PCL’ and ‘Electrospun’ at Title / Abstract), it is essential to assess the environmental degra- The test was performed according the standard guidelines dation of this new PCL architectural conformation. Degra- [23]. The activated sludge was collected from a sewage dation of collagen (used in the present study) in nature is treatment plant in Silkeborg (Denmark) at two instances also dependent on environmental conditions, which is well (April and August), and transported to the laboratory with known in forensic and archaeological science studying an adequate ventilation and temperature (20 °C ± 1). Once remains of humans and animals [13]. There are no studies in the laboratory, the sludge was sieved with a 2 mm mesh on the environmental fate of recombinant collagen. Herein, to calculate the total suspended solid concentration, which we aimed to determine the degradation of non-coated and was adjusted to 4000 mg/l with distilled water. An abiotic collagen-coated PCL electro-spun scaffolds in the envir- replicate of sludge was prepared for each sampling time by onment. Testing included the impact of sewage sludge and autoclaving it for 90 min at 121 °C and 15 psi. After cool- comparison of soils with different physico-chemical char- ing, pH of the abiotic was measured and adjusted to equal acteristics. To assess the potential biological impact of the that of the biologically active sludge. Biologically active produced scaffolds to soil invertebrates, the standard model and abiotic sludge (150 ml) were placed in 500 ml flasks, Enchytraeus crypticus [14–16] was used. and 6 pieces of 15 mg of test mesh were immersed indivi- dually inside a nylon mesh in each test vessel. The vessels were maintained with continuous shakes and a constant 2 Material and Methods temperature of 25 °C during the test. The meshes were removed after 3, 6, 11, 14, 21 and 24 days of exposure, 2.1 Test materials gently washed with distilled water and weighted (details ahead). Typical protocols for electro-spinning were utilised, as described previously [17–21]. In short, 200 mg/ml of PCL 2.3 Degradation test in soil (PC-12, Corbion, The Netherlands) were dissolved in 2,2,2- trifluoroethanol (TFE; 99.8%, Acros Organics, Ireland) and The degradation in soil was performed with three different was extruded at 100 µl/min from three syringes simulta- standard natural soils (Table 1), purchased from LUFA- neously through an 18 G stainless steel blunt needle (EFD Speyer (Germany). Test soils were dried for 24 h at 90 °C Nordson, Ireland). Upon application of high voltage and their moisture was adjusted to the 50% of the water (20 kV) between the needle and the collector (20 cm dis- holding capacity with a 1:1 mixture of deionized water and tance), the fibres were collected on a rotating mandrel (60 microbial substrate containing the native microbial and RPM). Three meshes were stacked together and compressed nematode community of the soils. The microbial substrate at Proxy Biomedical (Ireland) under optimal conditions of was based on 1 kg of test soil mixed with 2 l of deionized temperature / pressure. The compressed meshes were then water for 3 h, sieved through a 50 µm mesh and diluted 10 coated with 5 mg/ml human recombinant collagen isolated times [24, 25]. One vessel per soil type was prepared with form transgenic tobacco plants (hR Col; CollPlant®, Israel) 100 g of dried soil. Four pieces of ca. 10 mg of the test or bovine Achilles tendon collagen (BAT; Vornia Bioma- material (i.e., 1,000 mg mesh per kg dry weight) were then terials, Ireland). After coating, the meshes were allowed to placed in the soil individually inside a nylon mesh, and one air dry in a laminar flow hood at room temperature over- piece was removed after each respective sampling date: 7, night. The meshes were then cross-linked with 1-ethyl-3-[3- 14, 21 and 28 days of exposure. Journal of Materials Science: Materials in Medicine (2018) 29:51 Page 3 of 10 51 Table 1 Physico-chemical characteristics of LUFA 2.1, LUFA 2.2 and LUFA 2.4 soils including organic carbon content, pH and nitrogen content (average ± SD) and the soil type Soil parameter LUFA 2.1 LUFA 2.2 LUFA 2.4 Organic carbon (%) 0.71 ± 0.07 1.59 ± 0.13 2.03 ± 0.23 pH (0.01 M CaCl ) 4.9 ± 0.3 5.4 ± 0.2 7.3 ± 0.1 Nitrogen (%) 0.06 ± 0.01 0.17 ± 0.01 0.22 ± 0.02 Soil type Salty sand (uS) Loamy sand (lS) Clayey loam (tL) After this first experiment, a second was conducted to Afterwards, the remaining material was weighted in a evaluate the degradation of the electro-spun scaffolds in precision microbalance (0.001 ± 0.003 mg, Sartorius Micro soils amended with sludge. Sludge amendment was per- MC5-SC2). formed following the protocol of [26]. Briefly, 10 mg of To identify changes in surface area as the decomposition sludge was added to 100 g of dry soil, and after homo- progressed, it was attempted to use dye-adsorption/deso- genising, the soil moisture was adjusted to 50% of the rption techniques. However, since no consistent results were maximum water holding capacity (WHC) with deionized obtained for all the test materials, the method was dis- water. Parallel to this, a control (non-amended soil) was regarded. Scanning Electron Microscopy (SEM) was con- prepared by simply adjusting the moisture to 50% of the sidered, mainly providing qualitative structural indices, but WHC. Three pieces of 10 mg of the test material (i.e., not deemed to provide additional required quantitative 1000 mg mesh per kg dry weight) were then placed in the information, since the dry weight measures provided the soil individually, and one piece was removed at a time after required information i.e., disappearance of the PCL. 28, 56 and 180 days of exposure. 2.6 Data analysis 2.4 Toxicity test in soil One-way analysis of variance (ANOVA) followed by The test was performed according to the standard guidelines Dunnettʼs comparison post-hoc test (p ≤ 0.05) was used to of the Enchytraeid Reproduction test [14], using LUFA assess differences between control and treatments (Sigma- 2.2 soil and the test species Enchytraeus crypticus. Soil was Plot 11.0). Degradation Time (DTx) calculations were moist with deionized water adjusting the humidity to 50% performed modelling data to logistic sigmoid 2 parameters of the water holding capacity, and 25 g of moist soil was regression models, as indicated in Table 2, using the introduced in plastic vessels. The scaffolds were inserted in Toxicity Relationship Analysis Program (TRAP v1.22) each container, as explained above, in a concentration of software. 1000 mg per kg dry soil. Additionally, 6 control vessels were prepared containing 1000 mg/kg filter paper as control (VWR, filter paper No. 413). Organisms of synchronised 3 Results age (17-19 days) (for details see [15]) were transferred to each test vessel; 6 replicates were done. The exposure ran 3.1 Electro-spun scaffold characterisation for 28 days and was performed under constant temperature (20 °C) and a light regime of 16 h light/8 h dark. Water loss Microscopy analysis revealed no apparent differences and food (finely ground and autoclaved oat rolls) was between the groups in macro and micro level (Fig. 1). replenished weekly. Test endpoints include survival and Further, no significant difference (p > 0.05) in fibre diameter reproduction (number of juveniles). was observed between the groups (electro-spun alone: 2.2 ± 1.3 μm; coated with BAT Col I: 1.7 ± 0.8 μm; coated with 2.5 Determination of weight loss hR Col I: 2.1 ± 1.0 μm; n = 100 measurements). To determine weight in the above test without interference 3.2 Activated sludge exposure test from biofilm or soil particles attached to the surface of the test materials, a sonication method was used [27–29]. The The degradation rates were clearly much lower in the test materials were sonicated in deionized water for 2 min. abiotic conditions (Fig. 2). For the April sludge exposure, (Branson 5510, 50-60 Hz and 110 VAC, Fisher Scientific, abiotic conditions caused degradation of only 17% for PCL, US)todetachpossible biofilm from the electro-spun 4% for BAT and 16% for hR (21 days, 24 days was lost). scaffold, washed again and dried at 37 °C for 24 h. The exposure in biotic conditions caused a significant 51 Page 4 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:51 Table 2 Degradation time (days) for the materials tested in sludge collected in April (one value for the August test sludge for PCL hR), indicating the DT50 and DT90 (time to degrade 50% and 90%) estimates and respective model and parameters (Sigmoid 2 parameters; S: Slope; Y0: intercept) DT50 (days) DT90 (days) Model and parameters Biotic PCL 22 [18<CI<26] 33 Sigm. 2 param. S0=0,032, Y0=100; PCL (August) 116 [15<CI<896] – Sigm. 2 param. LogX S0=0,370; Y0=100 PCL hR 25 [21<CI<29] 37 Sigm. 2 param. S0=0,028; Y0=100; PCL hR (August) 125 [127<CI<376] 256 Sigm. 2 param. S0=0,0032; Y0=100; PCL BAT 20 [17<CI<23] 37 Sigm. 2 param. S0=0,035; Y0=100; Abiotic PCL n.e. n.e. – PCL hR 23 [20<CI<26] 29 Sigm. 2 param. S0=0,092; Y0=100; PCL BAT n.e. n.e. – CI 95% confidence interval Fig. 1 Stereo and scanning electron microscopy analysis revealed no apparent differences between the groups (PCL electro-spun scaffold alone (PCL) and PCL electro-spun scaffolds coated with BAT Col I (BAT) and rH Col I (hR)) Journal of Materials Science: Materials in Medicine (2018) 29:51 Page 5 of 10 51 B: August Fig. 2 Degradation represented A: April as weight loss (% initial weight) 100 100 of PCL electro-spun scaffold (PCL), PCL BAT electro-spun scaffold (BAT) and PCL hR 60 60 electro-spun scaffold (hR). a exposure from 0 to 24 days to activated sewage sludge (solid lines) and the abiotic sludge (dashed lines) collected in April. 0 0 b exposure from 0 to 180 days to 1 28 56 180 036 1114 2124 activated sewage sludge (solid time (days) time (days) lines) and the abiotic sludge (dashed lines) collected in PCL Bio hR Bio BAT Bio August PCL Ab hR Ab BAT Ab Fig. 3 Pictures of the various PCL electro-spun scaffolds at day 0 (up) and at day 24 (down) of exposure to the biotic sludge i.e., PCL, BAT and hR. Corner scale bar: 1 mm (P < 0.05) higher degradation of all materials during the 3.3 Degradation test in soil 24 days (Fig. 2): with weight loss around 50% i.e., measured 56% for BAT, 51% for PCL (51%) and 45% for hR. The The PCL electro-spun scaffolds degradation was generally time for degradation estimates can be depicted in Table 2. low, although hR showed a 20% weight loss after exposure The microscopic observations also illustrate a higher to LUFA 2.4 soil for 28 days (Fig. 4). In LUFA 2.1 and degradation on the PCL electro-spun scaffold (Fig. 3). LUFA 2.2 soils the degradation was below 6%. Fungal hyfa Regarding the long-term degradation of the electro-spun proliferation was observed in non-coated and coated PCL scaffolds tested in the sludge collected in August, it was electro-spun scaffolds in the three tested soils after 7 days of slower than in the experiment performed in April for all exposure to all the soils (Fig. 5). materials (Fig. 2). The weight loss of PCL electro-spun Longer exposures of PCL electro-spun scaffolds showed scaffold was similar for both sludge types, and concerning degradation on the first 28 days in the three soil treatments hR electro-spun scaffold, the degradation was 30% lower (microbial substrate, sludge-amended soil and soil). Sludge after 180 days in August sludge than after 28 days in April amendment caused an increased degradation of the PCL and sludge. hR compared to the soil in LUFA 2.1 soil, although BAT % start weight 51 Page 6 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:51 soil soil + microbial substrate soil + sludge amended Fig. 4 Degradation represented 100 100 100 as weight loss (% start weight) 90 90 of electro-spun scaffolds PCL, 80 80 80 hR, and BAT, in three soil types: LUFA 2.1, LUFA 2.2 and 70 70 70 LUFA 2.4. Left column: 60 60 LUFA 2.1 LUFA 2.1 LUFA 2.1 Exposure over 28 days to soils 07 14 21 28 028 56 180 028 56 180 with added microbial substrate. time (days) time (days) time (days) Middle column: Exposure over 180 days to soils (no soil + microbial substrate soil soil + sludge amended amendments). Right column: 100 100 Exposure over 180 days to soil 90 90 90 amended with August sewage 80 80 80 sludge. The regression lines to 70 70 70 the data are given per material 60 60 LUFA 2.2 60 LUFA 2.2 and soil in the table below, LUFA 2.2 showing the corresponding R 07 14 21 28 028 56 180 0 28 56 180 time (days) time (days) time (days) soil + sludge amended soil + microbial substrate soil 100 100 100 PCL 90 90 90 hR 80 80 BAT 70 70 70 60 60 60 LUFA 2.4 LUFA 2.4 LUFA 2.4 0 7 14 21 28 028 56 180 028 56 180 time (days) time (days) time (days) Soil Type BM soil + microbial substrate soil soil + sludge amended PCL -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 LUFA 2.1 hR -0,0138xdays+ 96,875, R² = 0,0042 -0,0138xdays+ 96,875, R² = 0,0042 -0,0138xdays+ 96,875, R² = 0,0042 BAT -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 PCL -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 LUFA 2.2 hR -0,0138xdays+ 96,875, R² =0,0042 -0,0138xdays+ 96,875, R² = 0,0042 -0,0138xdays+ 96,875, R² = 0,0042 BAT -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 PCL -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 -0,1823xdays + 99,346, R² = 0,7109 LUFA 2.4 hR -0,0138xdays+ 96,875, R² =0,0042 -0,0138xdays+ 96,875, R² =0,0042 -0,0138xdays+ 96,875, R² = 0,0042 BAT -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 -0,0969xdays + 99,634, R² = 0,5387 Fig. 5 Pictures of the PCL electro-spun (left column) (PCL), PCL electro-spun hR (central column) (hR), and PCL electro-spun BAT (left column) (BAT) after 28 days of exposure to LUFA 2.4 soil. Scale bar: 1 mm showed a similar degradation. Sludge amendment in LUFA 3.4 Toxicity test in soil 2.2 soil caused an increase in degradation only for BAT, while in LUFA 2.4, the degradation increased 20% for PCL No significant effects (p > 0.05) occurred for E. crypticus in and BAT (Fig. 4). terms of reproduction (Fig. 6). The degradation did not % start weight % start weight % start weight Journal of Materials Science: Materials in Medicine (2018) 29:51 Page 7 of 10 51 2.0 y=-0.0237x + 0.9732, R =0.0039 80 120 1.5 1.0 40 60 40 0.5 juveniles mesh 20 0.0 0 0 0 1234 0 PCL hR BAT weight loss (% initial weight) Test material Fig. 6 Results from the Enchytraeid Reproduction Test for Enchy- following this exposure (left numbers), and the correlation (or lack traeus crypticus exposed for 28 days in LUFA 2.2 soil to the PCL thereof) between the weight loss and the number of individual per electro-spun scaffold materials: PCL, hR and BAT. A: reproduction. adult B: the remaining weight (given as % of initial weight) of the mesh exceed the 2.2% of the initial weight during the 28 days of certain and unknown complexity in the process and that this the test (Fig. 6, left), and the number of juveniles per adult is not fully controlled or predictable. was not related to the degradation of the test materials (Fig. For the electro-spun scaffolds, the biodegradation during 6, right). the first 7 days did not differ between the non-coated and collagen-coated materials, but their degradation was slow probably due to the lower surface area (total or available to 4 Discussion microorganisms) in contact with the sludge, as the biotic degradation is known to occur at the surface [32]. As The present study showed the effect of the structure and the expected, the abiotic degradation was lower, and this has presence/absence and coating type of PCL materials on their been previously reported for sludge exposures [33], show- biodegradability and environmental fate. ing that decomposition was biological mediated. Degradation of PCL polymers by microorganisms in vitro The physico-chemical properties of soils are among the and in different environmental compartments has been well factors determining its microbial and fungal composition, studied, but the information of PCL is still very limited, which are the main decomposers of PCL. The differences particularly in regard to the differences between the electro- observed in biodegradation capacity related to soil type spun scaffold adaptations. Although biodegradation can be were expected although the exact combination of factors is defined by different parameter measurements such as loss of far less understood. For instance, pH is a decisive factor for mechanical properties (tensile strength, crystallization etc.), microbial enzymatic activity, since the enzymes require an CO production, morphological changes evaluated by SEM optimal pH for their correct functioning. Moreover, toler- or optical microscopy, etc [30]., the focus here was on ance to high or low pH is species/genus specific and, thus, physical disappearance, i.e., weight loss (dry weight), microbial diversity is strongly determined by pH, e.g., pH combined with optical microscopic observations to detect values below 5 decrease microbial decomposition and breakage or other types of changes in the surface. activity in soils [34, 35]. On the other hand, OC and N The current prospect is that the first biodegradation content represent growth-limiting resources and shape process occurs in sewage treatment plants, where industrial microbial community composition and activity [36, 37]. and domestic PCL discharges can be concentrated. As Accordingly, the decreasing gradient of pH, OC and N quantified here, the degradation of PCL materials is sludge- content of LUFA 2.4, 2.2 and 2.1, respectively, coincides dependent. In the present, the sludge was collected from the with the highest degradation measured in LUFA 2.4 soil, exact same sewage treatment plant at two time points, April followed by LUFA 2.2 and, finally, LUFA 2.1 soil. The and August, hence differences must be due to the varying presence of collagen coating in the PCL had an inhibiting waste water characteristics. This is not surprising and has effect on the degradability of the PCL mesh. This may be been previously observed regarding nonylphenol and because microbial collagenases are only produced after organic compound degradation [31]. In our study, the April exposure as a response, hence increasing the time needed sludge showed higher degradation efficiency than the for its degradation. On the other hand, although different August sludge. Additionally, the degradation rate was dif- bacteria genus commonly found in soils, such as Pseudo- ferent and the material-dependent degradation was also monas and Aeromonas, produce collagenase enzymes, it is distinct, e.g., the most degraded PCL material in April was not so common for soil fungi, and mainly nematophagous the least degraded in August. This indicates that there is a fungi species show collagenase activity. However, PCL No. juveniles (per adult, AV±SE) mesh weight (%) No. juveniles (per adult, AV±SE) 51 Page 8 of 10 Journal of Materials Science: Materials in Medicine (2018) 29:51 degradation by fungi is well known. The fungal coloniza- issues, such as the ones recommended for the testing of tion of the PCL materials observed during the test indicates nano-materials, e.g., the need for longer test duration (or that biodegradation was caused by fungi to some extent, and surrogates), are obviously in the same line for biomaterials. since fungal collagenase activity was not likely to occur in soil fungi, the collagen coating could prevent fungi from degrading the PCL. The observed weight loss of electro- 5 Conclusions spun scaffolds after 28 days of exposure is similar to the degradation of disc-shaped PCL pieces in soil after 35 days This is the first time that such biomaterials have been tested [38], i.e., 5% in a soil with 80% organic matter content. for environmental hazard. Biodegradation of the tested PCL These authors also reported the importance of the PCL materials was coating- and exposure-dependent. There was structure for the degradation, with the amorphous regions a clear faster biotic than abiotic degradation. The coated being the first ones to be degraded. electro-spun materials degraded as fast (or faster) then the Overall, the degradation of PCL materials in soil was non-coated PCL electro-spun. The time required for total much slower than in the activated sludge, rendering weight degradation exceeds the time period in sewage treatment loss values below 30% after 6 months of exposure in the plants, hence partial decomposition of PCL is expected best case, having a considerably long persistency (life time) when transfer takes place to amend soils. Soil types will in soil. In addition, the biodegradation is soil-type depen- determine differences between degradation efficiency. dent, so the persistence of PCL in soil can vary, depending Over-all the degradation patterns in the soil were similar, on the physico-chemical characteristics and microbial also for hr and BAT, except for the LUFA 2.4 soils. We community present in soils. For example, the presence of recommend the range of standard natural LUFA soils as a collagen-degrading microorganisms in soils is not ubiqui- good model basis to assess these variations so far. There are tous, and only 15% of organic horizons of spruce growth technical issues, like the test material form (mesh) or the test soil samples and 37% of garden soil samples showed duration, that deserve further consideration and optimiza- microbial collagenase activity [39]. In fact, according to tion for an adequate environmental hazard assessment of present results, the enrichment of soils with microorgan- biomaterials. isms from activated sewage sludge could increase the Acknowledgements This study was supported by funds from the degradation of the PCL electro-spun scaffolds in soils European Commission in the FP7 NMP project Green nano mesh (G. decreasing its persistency in soil, as in agreement with A. No. 263289). Further support was provided by BIORIMA H2020- previous studies which showed the beneficial effect of NMBP-2017 (GA No. 760928), by CESAM (UID/AMB/50017-POCI- sludge amendment in the microbial activity of soils [40–42] 01-0145-FEDER-007638) via FCT/MCTES through national funds (PIDDAC) and the co-funding by the FEDER, within the PT2020 and the consequent increased degradation efficiency. The Partnership Agreement and Compete 2020), and by Science Founda- presence of the PCL in soil did not affect E. crypticus,an tion Ireland, Career Development Award Programme (grant agreement invertebrate soil model representative, in terms of repro- number: 15/CDA/3629); Science Foundation Ireland and the European duction. Moreover, the degradation of the test PCL and the Regional Development Fund (grant agreement number: 13/RC/2073); EU H2020, ITN award, Tendon Therapy Train Project (grant agree- juvenile production were not correlated. We cannot exclude ment number: 676338). that this is due to the low degradation level within the test period and the test period itself, which is short compared to Compliance with ethical standards the material lifetime. The conclusion is similar to studies made with microbial substrates [43–47] where PCL was Conflict of interest The authors declare that they have no conflict of not toxic. interest. 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Published: Apr 30, 2018

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