J Mater Sci: Mater Med (2017)28:127 DOI 10.1007/s10856-017-5936-1

Original Research

BIOCOMPATIBILITY STUDIES

Influence of direct or indirect contact for the cytotoxicity and blood compatibility of spider silk J. W. Kuhbier1 V. Coger1 J. Mueller1 C. Liebsch1 F. Schlottmann1 V. Bucan1 P. M. Vogt1 S. Strauss1 ●

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Received: 6 February 2017 / Accepted: 21 June 2017 © Springer Science+Business Media, LLC 2017

Abstract Spider silk became one of the most-researched biomaterials in the last years due to its unique mechanical strength and most favourable chemical composition for tissue engineering purposes. However, standardized analysis of cytocompatibility is missing. Therefore, the aim of this study was to investigate hemolysis, cytotoxicity of native spider silk as well as influences on the cell culture medium. Changes of cell culture medium composition, osmolarity as well as glucose and lactate content were determined via ELISA measurement. Possible hemolysis and cytotoxicity in vitro of spider silk were performed via measurement of hemoglobin release of human red blood cells or relative metabolic activity of L929 fibroblasts, respectively, according to international standard procedures. In ELISA measurement, no significant changes in medium composition could be found in this study. Spider silk was not hemolytic in direct and indirect testing. However, a borderline cytotoxicity according to definitions was found in indirect cytotoxicity testing. Nevertheless, in direct cytotoxicity testing, relative metabolic activity measurement revealed that spider silk is not cytotoxic under these conditions. This is the first study to conduct standardized tests regarding cytotoxicity and hemolysis of native spider silk, which might be considered inert in cell culture. As neither hemolysis nor cytotoxicity was found in direct contact in standardized procedures, safety in biomedical applications may be assumed. The indirect cytotoxicity seems to play a

* J. W. Kuhbier [email protected] 1

Department of Plastic, Aesthetic, Hand and Reconstructive Surgery, Medical School Hannover, Carl-Neuberg-Strasse 1, Hannover 30625, Germany

minor role in vivo. However, a borderline toxicity was revealed, suggesting potential leachables not yet identified. Graphical Abstract Displays one of the weaving frames used in this study after seeding with the single drop technique described herein

1 Introduction A scaffold material for musculoskeletal tissue engineering should be applicable for bioengineering purposes, i.e. it should have a high biocompatibility, a degradation rate suitable for the desired tissue and the ability to guide ingrowth of surrounding cells to promote regeneration [1]. Hence no greater pH-changes and influence of microenvironment should occur, inflammation and foreign body reaction should be avoided or kept mild, and complete replacement of scaffold by the desired tissue grown de novo should take place slowly and stepwise to grant mechanical stability [2]. Synthetic polymers like polyglycolic acid (PGA) or polylactic acid (PLA) promise a good moldability, however, they often possess poor mechanical properties or unfavourable

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degradation behavior [3]. Hydrolysation processes, as they occur in rapid acidic degradation of PLA, alter the pH and thus decrease regeneration [4]. Collagen owns excellent mechanical properties and is decomposed in a neutral milieu enzymatically, but cross-linking is required to ensure longterm stability [5–7]. Additionally, immunogenicity causing allergic reactions has been described [6]. Other widely used biomaterials are decellularized allogenic or xenogenic scaffolds (e.g. AlloDerm® as allogenic human skin scaffold or Surgisis® as xenogenic porcine small intestinal submucosal scaffold) [8]. Likewise, a significant loss of stability during the degradation process could be proven for Surgisis® as well as high lymphocyte invasion was described [9]. Possible alternatives are silk-based biomaterials, which offer an excellent stability as well as slow enzymatic degradation [10, 11]. Nevertheless, the application of silkworm silk is controversial due to possible immunological intolerance [12–14]. Silkworm silk is composed of four proteins, i.e. the literal silk proteins heavy and light chain fibroin, an additional protein called P25 as well as the coating, the glue-like sericin, whereof sericin seems to be responsible for the immunogenicity [13, 14]. In the common purification processes, the removing of sericin may be incomplete resulting in an immune response [13]. In contrast, spider silk offers high bio- and cytocompatibility without further modification. Native spider dragline fibres could be utilized to guide fibroblast growth on miniature weaving frames [15]. In other studies, dissolved egg sac silk was moulded into porous scaffolds and chondrocyte growth as well as biocompatibility was investigated [16, 17]. In a rodent model, fibrotic and granulomatous response as well as giant cell invasion were observed, however spider silk scaffold showed less foreign body reaction than polyglactin sutures [16]. Another studies demonstrated that immunoreactions to dragline silk fibers implanted subcutaneously in pigs were comparable to or less than in established biomaterials [9, 18]. Additionally, spider silk holds unique mechanical purposes with very high tensile strength and breaking energy [19]. It is sterilizable due to its high temperature stability [15]. Therefore, the aim of the study presented here was to investigate cytotoxicity and hemolysis in vitro following international standard protocols. This is the first time that such standardized investigations are performed for native spider silk [20–23].

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frames as described earlier [15]. Egg sac silk was harvested from aside the webs of the spiders and cut in half; the eggs were removed manually with sterile forceps. Before sterilization, egg sacs were rinsed extensively with 70% ethanol. Both dragline silk and egg sacs were autoclaved for 15 min at 121 °C and 2 bar. Experimental groups were native dragline spider silk weaving frames (in all experiments) and native egg sacs (in the testing of cell culture medium changes), controls were well bottoms of either 6-well or 12-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland). To take account for the porosity of the spider silk weaving frames in contrast to the continuous area of the well bottoms, the area of the total weaving frames covered by spider silk fibres and thus available for cell attachment, i.e. the possible attachable area (PAA) of the weaving frame, was determined in two representative samples. It was calculated by taking digital photographies with a fluorescence microscope (Keyence BZ8000, Keyence Deutschland GmbH, Neu-Isenburg, Germany) and measuring the area darkened by the spider silk fibres with Image J software (Open Source software from url:http://imagej.net/ImageJ2). 2.2 Testing of changes in cell culture medium For testing of potential changes in cell culture medium composition, 6-well plates were prepared with either 1 mg of dragline silk or 5 mg egg sac silk. For cell culture, murine fibroblasts (NIH 3T3; LGC Standards GmbH, Wesel, Germany) were cultured in DMEM medium supplemented with 10% fetal calf serum (FCS), 1% sodium-pyruvate and 1% gentamycin solution (10.000 µg/ml; all by Biochrome; DMEM growth medium). Concentration of cells was set to 5 × 104 ml−1 and 200 µl of this suspension was carefully dripped onto the spider silk as described earlier [24]. After 1 h of adherence, 2 ml of DMEM growth medium were added. After 3 and 7 days, samples and medium were analyzed. Osmolarity was determined via freezing point measurement with an Osmometer (Micro Osmometer Type 13/13DR; H. Roebling Messtechnik GmbH, Berlin, Germany), determination of glucose and lactate concentrations were performed via ELISA with an YSI Select 2700 (YSI Incorporated, Yellow Springs, OH, USA). Median and standard deviation (SD) were calculated, student’s t-test was performed to analyse the difference between 3 day and 7 day values.

2 Materials and methods 2.3 Indirect and direct hemolysis testing of spider silk 2.1 Rearing of the silk, experimental design and measurement of weaving frames Dragline silk was collected from female spiders from the species Nephila edulis and reared on miniature weaving

To investigate the hemolytic potential of spider silk extracts and spider silk, their effect on the release of hemoglobin (Hb) from human red blood cells obtained from pooled blood from three human donors was investigated according

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Table 1 Specification of test items and conditions for direct and indirect hemolysis testing Test item

Spider silk

Weight-to-PBS volume ratio of 3 mg ml−1 (i.e. 2.5 m of a native spider dragline silk fiber with a mean weight of 0.0121 g + 0.0003)

Reference material nonhemolytic

RM-C (Hatano Research Institute)

Surface-to-PBS volume ratio volume ratio of 3 cm² ml–1

Positive control

Sterile Milli Q Synthesis ultrapure water

–

Negative control

PBS

–

Table 2 Specification of test items and conditions for indirect cytotoxicity testing Test item

Spider silk

Weight-to-extraction volume ratio of 4.84 mg ml−1 (i.e. 2.5 m of fiber with a mean weight of 0.0121 g)

Reference material cytotoxic

RM-A (Hatano Research Institute)

Surface-to-extraction volume ratio of 3 cm² ml−1

Reference material non-cytotoxic

RM-C (Hatano Research Institute)

Surface-to-extraction volume ratio of 3 cm² ml−1

Negative control

Aged complete growth medium (growth medium incubated under the same conditions as samples)

–

to ISO 10993-4:2009 and DIN EN ISO 10993.12:2012 as well as ASTM F756-12 [20, 22, 23]. Test items were prepared according to ASTM F756-12, specimen specifications are described in Table 1. All calculations for these tests were performed with MS Excel (Microsoft Deutschland GmbH). The hemolysis rate was further expressed in percent of the positive control (relative hemolysis). A hemolysis rate of more than 10% relative to the positive control was considered as a clear hemolytic effect according to ISO 10993-4:2009 and DIN EN ISO 10993.12:2012. 2.4 Indirect cytotoxicity testing of spider silk To investigate the influence of spider silk indirectly (i.e. via leachables like production residuals), a mouse fibrosarcoma cell line (L929; Leibniz Institute DSMZ, Braunschweig, Germany) was used according to the recommendations of ISO 10993-5.2009. Standard reference materials RM-A and RM-C (both purchased from Hatano Research Institue) were used as cytotoxic or non-cytotoxic reference, respectively. Aged complete growth medium served as negative control. For cytotoxicity assays, L929 cells were cultivated in growth medium containing RPMI 1640 medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS superior, Biochrom) and 1% CellCultureGuard™ (AppliChem GmbH, Darmstadt, Germany) at 37 °C, 5% CO2 and 90% relative humidity. Test preparation was performed according to ISO 1099312.2012 and ISO 10993-5.2009 [21, 22], specimen specifications are depicted in Table 2. Mean and SD were given in % of the negative control. According to DIN EN ISO

10993-5.2009, relative metabolic activity above 70% in comparison with the negative control suggests that the test item has no cytotoxic impact on the cells. A value below 70% suggests toxic leachables. 2.5 Efficacy of cell seeding and direct cytotoxicity testing Regarding the low values for metabolic activity of cells on the spider silk weaving frames in preliminary tests (data not shown), a poor efficacy of seeding was concluded. Therefore, a modified seeding technique (Single Drop seeding technique) based on the seeding technique described earlier [24] was developed and tested using CellTiterBlue® viability assay (CellTiter-Blue® Cell Viability Assay, Promega corporation, Madison, WI, USA). L929 cells were cultured in DMEM growth medium and were passaged at least once before use. For comparison of cells that actually attached to the spider silk compared to the controls (i.e. the well bottoms), an original seeding protocol was performed as follows: Weaving frames were put into 12-well cell culture plates (TPP) under a laminar flow sterile working bench and 25 µl of a solution with a cell density of 1 × 106 cells ml−1, i.e. an amount of 5 × 104 cells, were dripped onto the spider silk to allow cells adherence (Fig. 1). Another group contained samples seeded with the conventional technique, i.e. 5 × 104 cells were suspended directly in 2.025 ml medium and filled in the wells. After time periods according to the experimental design depicted in Table 3 (Tab. 3), wells were rinsed with PBS to wash off non-adherent cells and 1 ml of fresh medium with 200 µl CellTiter-Blue® was added, followed by incubation for

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another 90 min. In parallel, controls with weaving frames seeded without the above mentioned seeding technique as well as blanks filled with 1 ml of medium incubated under the same conditions were used for background controls. Test items and controls were performed in hexaplicates. From each well, 3 samples of 100 µl supernatant medium were transferred to a 96-well plate (96 Well Optical Btm Plt PolymerBase Black w/Lid; Thermo Fisher Scientific, Rochester, NY, USA) for measuring each well in triplicates.

Measuring with excitation and emission at a wavelength of 560 nm + 10 nm or 590 nm + 20 nm, respectively, was performed with a GENios microplate reader (Tecan Group Ltd., Maennedorf, Switzerland) using Magellan™ Data Analysis software (Tecan). Means were calculated and corrected by subtraction of the values of the corresponding background controls. Afterwards, SD was calculated for the corrected values with MS Excel. Mean and SD were given in % of the negative control. Testing for statistical significance was done with student’s t-test and the results were analyzed for variance with Analysis of Variance (ANOVA) followed by Bonferroni’s post-hoc correction to adjust for multiple comparisons.

3 Results 3.1 Geometry of weaving frames

Fig. 1 Displays osmolarity values for blank (Bln), blank + cells (Bln + Cells), egg sac silk w/o cells (Egg), egg sac silk with cells (Egg + Cells), dragline silk w/o cells (Sil) and dragline silk (Sil + Cells) on day 3 and 7. Values shown as mean + SD, no significances were found between the groups

Spider silk could be reeled on miniature weaving frames out of stainless steel in a regular cross-wise pattern. A discrepancy between the control groups and the weaving concerning the PAA was found. Due to the high porosity caused by the mesh pattern, a remarkable difference in the area to which cells can adhere to PAA was found. The mean + SD for the total area of the weaving frames was measured to 62.07 + 4.36 mm² with a PAASp of 31.94 + 3.89 mm², i.e. PAASp was 51.04% of the total weaving frame area.

Table 3 Displays experimental design of direct cytotoxicity test Group name

Treatment

Subgroup

Further treatment

Single 1.5 h

Seeding with single drop technique with 5 x 104 cells suspended in 25 µl medium on spider silk weaving frames. After 90 min of incubation, 2 ml of medium were added to each well, then separation into subgroups.

Spider silk fibres Well bottom

All wells were rinsed with PBS to wash off non-adherent cells and 1 ml of fresh medium with 200 µl CellTiter-Blue® was added, followed by incubation for another 90 min. From each well, 3 samples of 100 µl supernatant medium were measured.

Single 3 h

Seeding and incubation as above. Further incubation of 90 min, then separation into subgroups.

Spider silk fibres Well bottom

Conv 1.5 h

Seeding with the conventional technique, i.e. 5 x 104 cells were suspended directly in 2.025 ml medium and filled in the wells. After 90 min of incubation, separation into subgroups.

Spider silk fibres Well bottom

Conv 3 h

Seeding as above. After 180 min of incubation, separation into subgroups.

Spider silk fibres Well bottom

Control 1.5 h

Seeding with single drop technique with 5 x 104 cells suspended in 25 µl medium, but on the well bottom. Incubation for 90 min, then directly further treatment.

Control 3 h

Seeding as above. Incubation for 180 min, then directly further treatment.

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3.2 Changes in medium composition after incubation with spider silk Conditioned medium after incubation with spider silk was analyzed for changes in the chemical composition. There was no significant change in osmolarity over 7 days of incubation in either of the groups (Fig. 2a). In glucose concentration values, a decrease has been seen in all groups containing cells while an opposite progression could be seen in lactate measurement, indicating nutritional metabolic activity (Fig. 2 b+c). While glucose decrease and lactate increase were slightly higher in the blanks with cells compared to egg sacs with cells and dragline silk with cells, respectively, differences between day 3 and 7 were highly significant (p < 0.0001). No significant change could be seen in the control groups without cells. 3.3 Indirect and direct hemolysis testing of spider silk The baseline values (mean + SD) for the pooled blood were 0.639 + 0.036 mg/ml for PFH and 190 + 9 mg/ml for total blood Hb. Shortly after adding PBS to spider silk, the PBS solution showed a yellow but clear appearance. This did not change until the end of the extraction period. Likewise, supernatants (diluted plasma) of the spider silk samples appeared yellow colored. All other supernatant were colorless. In indirect hemolysis testing, supernatants from conditioned medium incubated together with spider silk were not hemolytic toward human erythrocytes. Concentration of free plasma Hb in spider silk extracts was 0.004 mg/ml + 0.007 mg/ml, a relative hemolysis of 0.37% + 0.63% was calculated (non-hemolytic reference material RM-C 1.46% + 0.37%); (see Fig. 3a). In direct hemolysis testing, spider silk surface was not haemolytic towards human erythrocytes. Concentration of free plasma Hb in spider silk was 0.056 mg/ml + 0.019 mg/ml, a relative hemolysis of 4.91% + SD 1.64% was calculated (non-hemolytic reference material RM-C 1.38% + 0.22%; see Fig. 3b). 3.4 Indirect and direct cytotoxicity testing of spider silk In indirect cytotoxicity testing, spider silk extracts were slightly cytotoxic to L929 cells. Relative metabolic activity (mean + SD) for spider silk was calculated to 65.9% + 8.7% (reference material RM-C 88.5% + 5.4%, reference material RM-A 0.3% + 0.4%; see Fig. 4). According to ISO 10993-5, it has to be considered as cytotoxic. To respect the porosity of the weaving frames, blank-corrected values of all weaving frame samples were further corrected using the correction factor of 0.5104 (i.e. the relative PAA of the weaving frames).

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With this correction factor, the true relative metabolic activity (SD were the same as above) were calculated to 93.36% for WF single drop 1.5 h, 71.18% for WF single drop 3 h, 45.07% for WF conventional 1.5 h and 40.64% for WF conventional 3 h, displaying values closer to the controls (p < 0.0001; Fig. 5).

4 Discussion In this study, the cytotoxicity and hemolysis effect of spider silk either in direct contact or indirectly via leachables was investigated. As the common seeding technique to fill a well with spider silk with a cell suspension appeared to be relatively ineffective, a modified seeding technique originally described by Wendt et al. was used to avoid rinsing of the cells from the fibres before they become adherent. 4.1 Consideration of the geometry of the weaving frames PAA was estimated by measuring the opaque area of the weaving frames. It was assumed that the measured PAA in the light microscopy equals the PAA for the cells as the silk fibres are the same barrier for the light as for the cells sedimenting down. By comparing the PAA to the total area of the weaving frames, the porosity of the weaving frames was determined and regarded as correction factor of 0.5104. It has to be considered that in the controls (i.e. the well bottoms), the PAA was significantly higher. However, as the same single drop seeding technique was used in the controls and thus the initial area to which the cells could adhere to was determined by the size of the drop on the well bottom. 4.2 Biocompatibility of silkworm and spider silk Silkworm silk of Bombyx mori is a United States Food and Drug Administration (FDA)-approved medical product and has also been proved as an excellent matrix for tissue engineering purposes in particular three-dimensional tissue engineering [11]. However, inflammatory reactions to virgin silk have been reported as well as granuloma foreign, i.e. foreign body reaction, or even cytotoxicity to silk sutures or just fibers in vitro and in vivo [12–14]. The coating protein sericin, which, according to common belief, is responsible for this reaction as “degumming”, i.e. removing of the sericin layer, causes less inflammation [12]. However, it was also observed that most degumming processes leave sericin residues that cause reactions. In contrast, no or just very mild inflammatory reactions were determined for spider silk [9, 16, 25].

127 Page 6 of 9 Fig. 2 Displays glucose (determined via ELISA a, determined via HLPC (c)) and actate (determined via ELISA (b)) concentration for blank (Bln), blank + cells (Bln + Cells), egg sac silk w/o cells (Egg), egg sac silk with cells (Egg + Cells), dragline silk w/o cells (Sil) and dragline silk (Sil + Cells) on day 3 and 7. Highly significant glucose decrease and lactate increase was seen in all groups (i.e. dragline silk, egg sacs and controls). Values shown as mean + SD, highly significant differences between groups (p < 0.0001) are marked with ***

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Fig. 3 Displays relative hemolysis in % of human erythrocytes after direct (a) or indirect (b) exposure to spider silk. After the incubation, the amount of plasma free hemoglobin was determined and hemolysis was calculated relative to the positive control, relative hemolysis values ≤ 10% relative to the positive control (black line) were

Attempts in genetically engineering of the main proteins of dragline spider silk resulted in yield of polypeptides containing multiple repetitions of small functional motifs instead of native-sized proteins [26]. For theses recombinant spider silk protein analogues, different biomedical applications have been tested. For example, spider silk protein analogues were used as a wound dressing for burn wounds and appeared highly biocompatible though biocompatibility was not measured qualitatively [27]. By binding different adherence motifs and glycopolymers either genetically or chemically to repetitive recombinant spider silk polyproteins, adhesion and proliferation of cells could be modulated [28]. By forming films, this functionalized spider silk polypeptide allowed formation of a keratinocyte monolayer. Additionally, human pluripotent Stem Cells, have successfully been cultured on two- and three-dimensional artificial spider silk matrices, indicating high cytocompatibility of recombinant spider silk [29]. Recombinant spider silk holds the advantage of large scale production capabilities and low tolerances in production. Nevertheless, it remains questionable if those genetically engineered protein analogues show the unique physicochemical properties of native spider silk as the five-layer-structure of the fiber is due to complex assembly of the silk proteins governed by

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considered as non-hemolytic. Both tests were performed in BioMedimplant Laboratory facilities (accredited according to ZLG-P359.09.05 and ZLG-AP-30710.30) and conducted according to Directive 93/42/EWG, 90/385/EWG and DIN EN ISO 17025

Fig. 4 Displays relative metabolic activity of L929 cells after 24 h of incubation with the extract of spider silk. The metabolic activity of the cells was determined after 24 h incubation. The positive control was set to 100%. RM-A (severely cytotoxic) and RM-C (non-cytotoxic) were used as reference materials, relative metabolic activity values ≤ 70% relative to the positive control (black line) were considered as cytotoxic. Test was performed in BioMedImplant Laboratory facilities (accredited according to ZLG-P-359.09.05 and ZLG-AP-30710.30) and conducted according to Directive 93/42/EWG, 90/385/EWG and DIN EN ISO 17025

ion gradients, shear forces and dehydration along the major ampullate gland [30, 31]. In the present study, native spider silk was chemically inert and did not alter osmolarity of cell culture medium

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Fig. 5 Displays metabolic activity of L929 cells after different time points of incubation in direct contact with spider silk as relative fluorescence (Control after 3 h was set 100%). Different seeding techniques were applied, i.e. the single drop technique and the conventional technique (see text for detailed description). Single 3 or 1.5 h = seeding with single drop technique and incubation for either 3 or 1.5 h, then separation of weaving frame with spider silk fibres and well

bottom. Conv 3 h or 1.5 h = seeding with conventional technique and incubation for either 3 or 1.5 h, then separation of weaving frame with spider silk fibres and well bottom. Control 3 or 1.5 h = seeding with single drop technique on well bottom without weaving frame with spider silk fibres and incubation for 3 or 1.5 h. All comparisons between groups showed a significance level of p < 0.05 except those marked as not significant (n.s.)

while nutritional metabolic activity of cells could be verified by time-dependent changes of glucose and lactate concentrations. No hemolysis could be measured either with indirect or direct contact. In indirect cytotoxicity measuring, the relative metabolic activity of the cultured cells was <70%, connoting cytotoxicity. A change in the colour of PBS to a shade of yellow after adding spider silk could be seen, indicating probable leachables on a basis of phosphorus, which might be specific to the golden-coloured spider silks like those from Nephila spp. [14]. Therefore, the presence of leachables might be proposed that are responsible for the indirect cytotoxicity. Comparing this finding to literature, it was reported that culturing endothelial cells with indirect contact to spider silk decreased proliferation compared to culturing cells in cell culture medium alone [32]. However, decrease of proliferation was much stronger in samples cultured with silkworm silk In a most recent study by Steins et al., conditioned medium showed no adverse effects on cell growth, thus the possible toxic effects may be questioned [33]. The investigation of spider silk in direct contact with cells with respect to the porosity of the weaving frames and thus the correction factor revealed that it was not cytotoxic. For the single drop technique both 3 and 1.5 h values were >70%, thus spider silk has to be considered as not cytotoxic in direct testing according to ISO 10993-5 [21]. Additionally, values for weaving frames and well bottom combined were higher than in the controls, further indicating that no

cytotoxicity exists in direct contact (Fig. 5). Considering the current state of research, this alteration of cell growth in indirect contact turns out to play a minor role in vitro and in vivo as no adverse effect of spider silk could be proven and spider silk displayed an excellent cyto- and biocompatibility [9, 15–18, 24, 25, 33].

5 Conclusion Native spider silk displayed low immunogenicity, high adhesion of cells and the capability of building functional tissues in previous studies. Most interestingly, spider silk promoted survival and engraftment of different cell types of entodermal [33], ectodermal [24, 25] and mesodermal [9, 15, 17] origin. Taken together, spider silk in direct contact to cells appears to be chemically inert and neither hemolytic nor cytotoxic in cell culture. It can be assumed that it is well suited as a scaffold for biomedical purposes, while in contrast indirect contact seems to affect cell growth and metabolism negatively. Hence, the indirect cytotoxicity seems to play a minor effect for biocompatibility in vivo as most studies suggest. Acknowledgements This article is dedicated to Kerstin Reimers. Parts of the study were funded by the Fritz-Behrens-Stiftung. Tests “Indirect cytotoxicity testing of spider silk” and “Indirect and direct hemolysis testing of spider silk” were performed in BioMedimplant Laboratory facilities (accredited according to ZLG-P-359.09.05 and

J Mater Sci: Mater Med (2017)28:127 ZLG-AP-30710.30) and conducted according to Directive 93/42/ EWG, 90/385/EWG and DIN EN ISO 17025. We hereby want to thank BioMedimplant for their kind support. An official approval for using the data is present. Compliance with ethical standards Conflict of interest interests.

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The authors declare that they have no competing 19.

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