Preliminary investigations of spider silk in wounds in vivo — Implications for an innovative wound dressing

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Preliminary investigations of spider silk in wounds in vivo — Implications for an innovative wound dressing Christina Liebsch a, Vesna Bucan a, Bjoern Menger a, Franziska Köhne a , Karl-Heinz Waldmann b , Desiree Vaslaitis a , Peter M. Vogt a, Sarah Strauss a, Joern W. Kuhbier a, * a

Department of Plastic, Aesthetic, Hand and Reconstructive Surgery, Medical School Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany b Clinic for Swine, Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany

article info

abstract

Article history:

The ideal wound dressing in particular for burn wounds has not been found yet. The aim of

Accepted 22 March 2018

this study was to investigate native spider silk as a novel wound dressing.

Available online xxx

Release of inflammatory cytokines of macrophages and neutrophile granulocytes was determined via ELISA after exposure to spider silk. Migration of dermal cells as well as

Keywords: Wound dressing Spider silk Wound healing Bio compatibility Biomaterial

angiogenesis on spider silk was visualized with live video microscopy or chorioallantois membrane model, respectively. Native spider silk was placed in full-thickness skin wounds in a sheep in vivo-model and wounds were evaluated after 2, 4, 6, and 8weeks histologically as well as per quantitative real-time PCR. Minimal inflammatory cytokine release could be seen for spider silk. Ingrowth of single capillaries into bundles of spider silk and migration of keratinocytes as well as fibroblasts on spider silk fibres was proven. Macroscopically, a comparable wound closure could be seen in spider silk and in sham controls. In histological evaluation, a thicker epidermis was observed in spider silk treated wounds while collagen III/I expression ratio was comparable in both groups. As native spider silk has been described as highly biocompatible, it might represent an innovative alternative to common wound dressings. © 2018 Elsevier Ltd and ISBI. All rights reserved.

1.

Introduction

A wound is considered to be the result of ‘disruption of normal anatomic structure and function’ [1]. Chronic or nonhealing wounds are open wounds that do not epithelialize and ultimately close within 3months. Another definition for

a chronic wound is a wound that has not shown a 20–40% reduction in area after 2–4 weeks of optimal therapy [2]. A chronic wound has to be considered as bacterially contaminated, while on the other hand bacterial contamination of the wound results in clinical infection and delays healing if more than 105 organisms per gram of tissue are present [3]. Additionally, to avoid hypertrophic scarring the timing until

* Corresponding author at: Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail address: [email protected] (J.W. Kuhbier). https://doi.org/10.1016/j.burns.2018.03.016 0305-4179/© 2018 Elsevier Ltd and ISBI. All rights reserved.

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wound closure should be less than 21 days [4]. Therefore, an early functional wound closure is mandatory. The gold standard for wound closure is the usage of split-thickness autografts, while still, after deep wound excision, elasticity and movability remain poor due to a missing sliding layer. Unstable scars with a risk for secondary wound healing deficits might result, indicating a need for functional skin coverage. Therefore, dermal matrices like IntegraTM (Integra LifeSciences Corp., Plainsboro, NJ, USA) are used to obtain a dermal layer beneath the transplanted split thickness grafts [5]. In these matrices, the idea is to support cellular ingrowth of dermal cells, i.e. to provide a scaffold instead of just optimizing the milieu for natural wound healing like modern wound coverages. These scaffolds may provide a matrix for reepithelialisation and support the ingrowth of new vessels. Other options include the use of decellularized dermal or amniotic matrices, either xenogenic or allogenic, which led to a significant reduction of wound area [6,7]. An ideal scaffold must be biomimetic, physically stable for implantation, biodegradable after in vivo implantation and should not be toxic for cells to replace or repair the original tissue or organ [8]. A material for such a scaffold may be spider silk. While previous studies investigate repetitive polypeptide analogues of spider silk proteins in wound healing, this is the first study using native spider silk fibres as a wound dressing. Aim of this study was to investigate in vitro immunogenic response to native spider silk to evaluate potential influences on the inflammatory phase of wound healing in terms of chemotactic response. Additionally, cell migration on native spider silk fibers was monitored. As a proof of concept, full-thickness skin wounds on the dorsum of sheep were set and spider silk was placed on the wound bed. Wounds were evaluated clinically and histologically as well as per quantitative real-time PCR (RT-PCR).

2.

Theory

The idea of using spider silk as a wound dressing dates back to the ancient roman medicine. In a comprehensive review, Newman and Newman presented historic medicinal applications of spider silk [9]. Many applications were dermatologic, both as styptic and therapy for skin lesions. However, these ancient applications were empiric and not based on basic research. In more recent investigations, there is evidence for excellent biocompatibility of spider silk [10–22]. Concerning dermal applications, a two-layer skin equivalent could be engineered using spider silk scaffolds [21]. Nevertheless, wound healing studies investigating in vivo implantation are missing. Therefore, the idea of this study was to study the applicability of native spider silk as a wound dressing in an in vivo approach and define the compatibility in an organism as well as analyse immunological response in vitro.

3.

Materials and methods

Promyelocytic leukemia cells (HL-60) were a kind gift from the Institute of Pharmacology of the Medical School

Hannover, Germany and cultivated in RPMI 1640 medium supplemented with 10% heat-inactivated Donor Horse Serum, 50 U/ml Penicillin/Streptomycin and 1% nonessential amino acids (all purchased by Biochrom AG, Berlin, Germany). Differentiation to granulocytes was induced by adding 1,25% DMSO to the culture medium for 5days, macrophage-like cells were established by addition of 800 nM Phorbol 12-myristate 1-acetat (Sigma-Aldrich, Munich, Germany) for 3days. Both cell types were incubated with native spider silk or polypropylene-polyglecaprone-251 composite (Ultrapro ; Ethicon, Norderstedt, Germany) for 4h, 8h, 24h, and 48 h (only macrophages). Both meshes were put onto 6-well-plates, filled with 4ml suspension of serum-free medium and 5
105 cells/ml. As positive control, cells were stimulated by 100ng/ml Lipopolysaccharides from Pseudomonas aeruginosa (LPS; Sigma). Cells cultivated on plastic served as negative control. Each group was tested thrice. After the different incubation periods, medium supernatants were collected and immediately tested with 1 ELISA DuoSets (R&D Systems, Wiesbaden-Nordenstadt, Germany) for human TNFa, IL1b and IL-6 concentration measurement according to the data sheet. An 8-fold standard dilution series was created to generate a standard curve. Measuring of absorption at a wavelength of 450nm10nm was performed with GENios microplate reader (Tecan Group Ltd., Maennedorf, Switzerland) using MagellanTM Data Analysis software (Tecan). Extinction values at 570nm were subtracted to eliminate potential background error. Each probe was analyzed in triplets; means and standard deviations (SD) were calculated with Microsoft Office Excel 2003. When Kolmogorov–Smirnov-test showed a normal distribution of data, a one-way Analysis of Variance (ANOVA) was performed followed by Bonferroni post-hoc-tests. Dragline silk was collected from Nephila edulis and reared on miniature weaving frames as described earlier [12]. Human adult calcium high temperature keratinocytes (HaCaTs) were cultured in DMEM/Hams F12 supplemented with 1% FCS, 1mM Sodium-pyruvate (PAA) and 0,2mg/ml Gentamycin solution (Biochrom AG) and seeded on silk frames by placing 100 ml cell suspension containing 1
106 HaCaTs onto the middle of the frame. To promote cell attachment on the silk fibres, plates were placed into an incubator at 37  C with 95% humidity/5% CO2 for 30min, later filled up with 2ml medium and then transferred to a chamber heated to 37  C of the Live Video Microscope (Keyence VHX-5000, Keyence Deutschland GmbH, Neu-Isenburg, Germany). To prove angiogenesis into a matrix of spider silk, bundles of spider silk were applied onto the chorioallantoic membrane (CAM) of fertilized chicken eggs, CAM preparation was performed as described earlier [23]. Eggs were incubated at 37  C for 3days, the eggshell was opened and spider silk applied to the CAM, followed by further incubation for 9days. Hereafter, CAM was extracted and fixed for paraffin embedding. 5 mm sections were stained with hematoxylin-eosin (H&E) and Masson trichrome. Microscopical observation was performed with an Olympus CK40 inverse microscope with software Cell^D (Olympus Deutschland GmbH, Hamburg). All animal experiments were performed with the approval of the Lower Saxony State Office for Consumer Protection and

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Food Safety (file number: 15/1755) according to the German Animal Welfare Law and the Directives of the European Union. Surgical procedures were performed in general anesthesia with isoflurane on adult sheep (n=4) followed by pain treatment with meloxicam for the following five days. Bilateral paravertebral wounds were set as rectangular skin incisions in two rows of three wounds on each side (Fig. 1a). Wounds on the left side were treated with spider silk, the right side remained untreated as Sham. Gaze wound dressings were applied to protect the wound sides. One row of three identical wounds with an area of 4cm2 served for biopsies after 2, 4 and 6weeks, additionally 2mm punch biopsies were taken in a standardized matter from the border region of the wound. In the second row, different wound sizes were set with an area of either 1, 4, or 9cm2. Dragline silk was collected and reared on a device of 30cm mounted on a motorized drum as described earlier [24]. Spider silk bundles with up to 500 fibers were autoclaved at 121  C and applied into the wounds by distributing them with two forceps (Fig. 1b). Native areas of skin served as controls. After 8weeks, all animals were sacrificed by pentobarbital 1 injection (Release , WdT eG, Germany). The wounds were explanted with all layers from muscle to skin and divided longitudinally for either histologic or RNA analysis. For histological analysis, specimens were cut into pieces and fixed, embedded, sectioned and stained with H&Eand Masson Trichrome. Stained sections were examined with an inverse light microscope (Olympus CKX41; Olympus, Hamburg, Germany). Thickness of epidermis was evaluated with Image J software in quintuplicates in each of the 4 animals

3

(Open Source software from http://imagej.net/ImageJ2). Total RNA was isolated from samples using the NucleoSpin RNAII Kit (MN Macherey-Nagel, Duren, Germany), RNA concentration was measured by photometry at 260nm. The quality of total RNA was verified by the integrity of 18S/28S ribosomal RNA in 1% ethidium bromide-stained agarose gels. Reverse transcription was performed with 1 mg total RNA using iScriptTMcDNA Kit (Bio-Rad Laboratories, Hercules, CA). RT-PCR reaction was carried out in 15 ml samples with 5ng cDNA and 10pmol of each forward and reverse primer (Table 1) and 2x SYBR green Sensi-Mix DNA Kit (Quantace, London, U.K.). Relative gene expression was determined by the fluorescence intensity ratio of the target gene to Cytochrome C (CYC). The initial denaturation step at 94  C for 4min was followed by 40 cycles of denaturation for 30s, annealing for 30s at 65  C, extension at 72  C for 1min, and final extension step at 72  C for 10min. All experiments of RNA measurement were carried out in triplicates and repeated at least at three independent times. The specificity of the Q-PCR products was proven by the appropriate melting curves. Results were evaluated with qBasePLUS software (Biogazelle, Zwijnaarde, Belgium). Calibrated normalized relative quantity values were exported. Singular values for Coll A1, Coll 3A1, ASMA, and CytC were determined and ratios for Coll A1/Coll 3A1 as well as either Coll A1, Coll 3A1 or ASMA to CytC calculated. For all results from the in vivo-study, testing for statistical significance was done with student's t-test and the results were analyzed for variance with one-way ANOVA followed by Bonferroni post-hoc-tests.

Fig. 1 – Depiction of wound design and spider silk application as well as wound appearance after 8weeks. A: Design of the paravertebral wounds in the sheep model. One row of wounds with an area of 4cm2 served for biopsies after 2, 4 and 6weeks, in the second row, different wound sizes were set with an area of either 1, 4, or 9cm2. B: Application of spider silk in the wound with forceps. C, D: Representative sample of wound appearance after 8weeks before explantation; C: spider silk group, D: sham operation group. Please cite this article in press as: C. Liebsch, et al., Preliminary investigations of spider silk in wounds in vivo — Implications for an innovative wound dressing, Burns (2018), https://doi.org/10.1016/j.burns.2018.03.016

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Table 1 – Forward and reverse primers used in RT-PCR. Primers were designed based on information from the genomic data base. CYC: Cytochrome C. ColA1: Collagen A1. Col3A1: Collagen 3A1. ASMA: Alpha-smooth muscle actin. Target gene CYC Coll A1 Coll3A1 ASMA

4.

Direction of primer Forward Reverse Forward Reverse Forward Reverse Forward Reverse

Base sequence 0

5 - CCC ACC GTG TTC TTC GAC AT -30 50 - CCA GTG CTC AGA GCA CGA AA -30 50 - TCG CTG GCA TTG GAG GAG AAA AAG -30 50 - GGG AAC CGT CGG GAC TAA TGA GG -30 50 -CAT CAG CAA GAA CCC CAA GGA CAA-3 50 -GGT GGA CAT CAG GCG CAG GAA GGT-30 50 - TGA CCG CAT GCA GAA AGA GA -30 50 - GAG CCG CCA ATC CAG ACA -30

1

Results 1

Immunogenic testing of spider silk and Ultrapro meshes in vitro showed no significantly increased values of cytokine liberation in granulocyte or macrophage cell lines (Fig. 2a–d). The reaction to LPS was positive and showed typical timedependent liberation depending on the cytokine and cell type while in the granulocyte culture, a liberation of IL1b after 24h 1 could be seen for Ultrapro (Fig. 2b). In all other granulocyte assays and time points, liberation of cytokines after exposure 1 to spider silk or UItrapro was extremely mild compared to LPS (p<0.001). In macrophages, a delayed mild liberation of TNFa

could be seen in granulocytes for spider silk and Ultrapro compared to LPS (p<0.05 after 4h, at all other time points p<0.001). Cell migration of a co-culture of HaCATs on spider silk meshes was verified by video microscopy (Supplementary Video S1 in the online version at DOI: 10.1016/j.burns.2018. 03.016). In the CAM assay, capillaries sprouted in spider silk bundles indicating neovascularization (Fig. 3a). Spider silk fibres could be observed as double refractory structures (white arrows in Fig. 3b and c) in histologic sections. Capillary bundles were in close proximity to spider silk fibres (black circles in Fig. 3b and c), thus it can be considered that spider silk favours

Fig. 2 – Diagram of time-dependent cytokine liberation of cell culture of granulocytes and macrophages together with spider 1 silk, Ultrapro and Lipopolysaccharide (LPS) determined by ELISA. A: Liberation of TNFa in granulocyte culture. B: Liberation of IL1b in granulocyte culture. C: Liberation of IL6 in granulocyte culture. D: Liberation of TNFa in macrophages. *P<0.05, ***P<0.001. Please cite this article in press as: C. Liebsch, et al., Preliminary investigations of spider silk in wounds in vivo — Implications for an innovative wound dressing, Burns (2018), https://doi.org/10.1016/j.burns.2018.03.016

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neovascularization or at least is not disadvantageous for blood vessel growth. Macroscopically, all wounds in both groups in the in vivo experiments were closed after 8weeks with no significant differences, though there was more scab apposition in shams (Fig. 1c and d). Tendentially, shams showed a padded scar formation as well as mild invasion of inflammatory cells into the superficial dermis. In the histological sections, spider silk fibres could be observed in biopsies after 2weeks but only in a few samples after 4weeks. After 6weeks, spider silk fibres occured only occasionally. Histologic staining revealed an overall thicker epidermis in the spider silk group compared to shams (Fig. 4a and b). Epidermis and dermis formation could be observed, in spider silk with melanocytes in the basal cell layer of the epidermis (black circles in Fig. 4a). There appeared to be a trend for the epidermal measurements to be thicker than in sham operations all wound areas and all time points, though only significant between the spider silk 9cm2 group after 2weeks to the controls in week 2, between the sham group 9cm2 after 2weeks and the controls in week 2 as well as between spider silk and sham group 9cm2 after 2weeks, respectively (p<0.05; Fig. 4c). In all other time points or wound sizes, spider silk showed tendentially thicker epidermis, but differences were not significant. In mRNA analysis, relative expression of CollA1, Coll3A1 and ASMA was determined. In both CollA1 and Coll3A1, spider silk displayed tendentially higher values than conventional wound dressings though not significantly. Both groups showed significantly higher expression for CollA1 (p<0.05; Fig. 5a) and Coll3A1 (p<0.01; Fig. 5b), respectively. Relative gene expression for ASMA was highest in conventional wound dressing, though not significantly (Fig. 5c), indicating tendentially higher wound contraction. Additionally, ratio of mRNA expression of CollA1 vs Coll3A1 (Coll A1/Coll3A1 ratio) was calculated to 0.680.12 for spider silk, 0.800.32 for shams and 0.670.08 for native controls (p>0.05 between all groups; Table 2).

5.

Fig. 3 – Microscopy of spider silk in the chorioallantois membrane. A: Light microscopy without staining of a capillary between spider silk fibres. B: HE staining of a capillary (black circle) between double refractory spider silk fibres (white arrows). C: Masson trichrome staining of two capillaries (black circles) between double refractory spider silk fibres (white arrows).

Discussion

Spider silk displayed minimal immune response, comparable 1 to Ultrapro except expression of IL1b after 24h, which was 1 nearly as high for Ultrapro as in the LPS group. IL1b and TNFa have been described to be the most potent cytokines in mediating inflammatory reactions and to play a part in chronic inflammation and activation of the adaptive immune system [25]. Additionally, IL1b has been described to direct macrophages to a dermal wound site [26]. In general, these findings are in line with Schäfer-Nolte et al. who found highest values 1 for RNA expression of TNFa were found in Ultrapro 4weeks after implantation and of IL1b 14days after implantation in rats [17]. Nevertheless, in that study IL6 expression was highest in spider silk group after 4days, but higher for 1 1 Ultrapro and for the xenograft matrix Surgisis 14days and 4weeks after implantation. Spider silk induces a mild foreign body reaction (FBR) with formation of Langerhans giant cells 1 though less than to polyglactin (Vicryl ) sutures [10,11,17]. This is in line with the findings of the in vitro-immunoassay herein, which suggest a mild activation of mononuclear cells.

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Fig. 4 – Microscopy of biopsy samples after 4weeks of 2cm2 wounds of both groups in comparison in H&E staining, bar represents 100 mm and measurement of epidermis thickness. A: Spider silk group. Black circles depict melanocytes. B: Sham operation group. C: Thickness of epidermis in mm depicted as mean+standard mean error (SEM). *P<0.05.

Altogether, the findings indicate an immune response at least 1 comparable to Ultrapro with no activation of the “first response” of the innate immune system. In live video microscopy, cells could be seen migrating along spider silk fibres in terms of a guided growth which was postulated hitherto (Supplementary Video S1 in the online version, at DOI: 10.1016/j.burns.2018.03.016) [10]. The importance of the fibrin clot and the fibrin network for migration of keratinocytes and thus reepithelialisation has been described [2,26,27]. It might be assumed that spider silk fibres take a role similar to that network and thus might favour wound healing.

In the CAM assay, capillary ingrowth could be observed between spider silk fibres, indicating neovascularization in spider silk scaffolds. The importance of angiogenesis for wound healing in particular in chronic wounds has been shown [28]. The ingrowth of capillaries into spider silk bundles was at least not hindered by chemotactic effects, nevertheless, as no quantitative assessment was performed, no statement can be made concerning possible alterations or comparisons to other materials. The sheep model has been described to be an adequate model for wound healing studies and allows the creation of a

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Fig. 5 – Relative levels of mRNA as determined by real-time polymerase chain reaction (RT-PCR) analysis. A: Ratio of mRNA expression (mRNA Ratio) of CollA1 vs CYC; B: mRNA Ratio of Col3A1 vs CYC; C: mRNA Ratio of ASMA vs CYC. Data are the means +SD for triplicate determinations which were repeated in three separate experiments. *P<0.05, **P<0.01 vs. control.

Table 2 – Ratio of mRNA expression of CollA1 vs Coll3A1 (Coll A1/Coll3A1 ratio)Standard deviation (SD) for the three groups in the experiments as well as p-value between the tests.

CollA1/Coll3A1 ratio SD

Spider silk vs. Control p = 0.9671

Spider silk

Sham

0.68 0.12

0.80 0.32

0.67  0.08

Sham vs. Control

Spider silk vs. Sham

p =0.5302

p =0.5573

relatively shallow wound with large surface area that is typical for wounds after traumatic injury (for example burn injuries after excision) or decubitus ulcers [29]. While there are previous studies existing that utilizes repetitive polypeptide analogues of spider silk proteins (so-called “recombinant spider silk”) as a superficial wound dressing in burn wounds, this is the first study to investigate native spider silk as a wound dressing [30]. In the study by Baoyong et al., the groups with recombinant spider silk showed faster healing in terms of healed wound area compared to collagen and saline.

5.1.

Control

Markers of hypertrophic scarring

A study of Schäfter-Nolte et al. found superior scar quality and less seroma formation in controls and shams [17]. In contrast, in the study at hand values for CollA1 and Coll3A1 were higher in spider silk and sham groups while CollA1/Coll3A1 ratio was lower than in controls, indicating immature scar tissue (Fig. 5). This is in line with the study investigating the repetitive peptide motif from spider silk by Baoyong et al., which found a higher hydroxyproline amount in the spider silk groups [30]. Spider silk displayed a higher ratio than the sham group, although not significantly, while the absolute values for CollA1 and Coll3A1 compared to the reference gene CytC differed significantly to the benefit of spider silk. Collagen production is an important function of activated myofibroblasts, aiding in

normal wound healing and contributing to enhanced tissue stiffness in fibrosis development. In the early phases of wound healing, Collagen III is expressed as a more unorganized prototype to cover the missing area of skin [31]. The measured values indicate a higher organization in the spider silk group than in the sham group, though not significantly and less than in controls. As the controls were biopsies from healthy skin, the finding is therefore not surprising. However, the values for the collagen ratio were higher in other studies investigating wound healing in sheep, which found a ratio of 3.33 for adult sheep after 4weeks [32]. In contrast, a newer study investigating calcium alginate as wound dressing found values for the CollA1/Coll3A1 ratio comparable to the findings of the study [31]. Albeit, that study used a rodent instead of a sheep model. Nevertheless, as CollA1/Coll3A1 ratio in our controls was comparable to the spider silk as well as the sham group, therefore we considered our findings as valid. Concerning ASMA expression, highest values were found in the sham, indicating higher rates of myofibroblasts and thus an earlier phase of wound healing (i.e. wound contraction) than in the spider silk group. Interestingly, melanocytes could be observed in spider silk, but not in shams. Melanocytes have been described to regulate keratinocyte and fibroblast contraction during wound healing and thereby formation of hypertrophic scars in vitro [33]. However, regarding the increased incidence of keloids in

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individuals with darker skin on the one hand and the dyspigmentation as a common problem in burn wounds and scars on the other hand, the role of melanocytes in vivo remains questionable [34].

5.2.

Biocompatibility of spider silk

Spider silk owns an excellent cyto- and biocompatibility that has extensively been described [10–22]. However, indirect contact to spider silk decreased proliferation of endothelial cells compared to cell culture medium alone, probably due to yet unknown leachables [18]. In contrast, conditioned medium after exposure to spider silk displayed no adverse effects on cell growth [16]. No adverse effects were seen in vivo [13–15,17,19–21] A constant degradation of spider silk fibres could be observed over the time course with almost complete disappearance after 8weeks. These findings are in line with a study that showed beginning degradation of epifascially implanted spider silk meshes after 4weeks [17]. After 4months, no spider silk fibres could be found in this study histologically. Spider silk meshes and, during the course of the study, the remaining scar showed mechanical stability while silkworm silk displayed a loss of tensile strength of 73% after 1month [35]. The same degradation kinetics were described by Vollrath et al. after subcutaneous implantation of dragline fibres in rats [11]. In contrast, a study investigating subcutaneous spider silk cocoon fibre implantation in a rodent model reported persistent fibres after 7weeks [10]. However, it has to be considered that cocoon fibres are produced in the tubuliforme gland instead of the major ampullate gland and thus possess another protein composition [36]. Due to incomplete understanding of spider silk formation and its control mechanisms, recent attempts to engineer recombinant spider silk lack active domains [37]. However, biotechnological production holds the advantage that spider silk proteins can be functionalized, for example to improve adhesion of cells or antimicrobial activity [38–40]. The importance of growth factors to optimize wound healing has been shown and the usage of wound dressings as drug delivery system has been suggested [41]. So it might be a strategy to couple recombinant spider silk with growth factors or use it as drug delivery matrix [42]. This might give implications to address difficulties of chronic wounds like chronic inflammation, fibrosis and senescent fibroblasts might alter the use of spider silk

5.3.

Limitations of the study

This study displays a first preliminary investigation of the use of native spider silk as an innovative wound dressing. More frequent measuring of the wound areas were not performed to minimize stress to the animals due to frequent shavings of the wound. Therefore, no statement whether spider silk accelerated wound closure time or not is possible. Likewise, quantitative measurement of vascularization is missing in the CAM model. Neither the dynamic interplay between dermis and epidermis concerning formation of hypertrophic scars nor the formation of neocollagenesis could be monitored by the design of this pilot study. As an acute wound model was investigated, no statements can be made regarding the difficulties of chronic wounds.

6.

Conclusion

Summarizing, spider silk can been considered as eligible for application as a dressing for acute wounds. The ancient use of spider silk could be confirmed with modern basic research methods. No adverse reactions were seen in terms of inflammation or delay of wound healing. Spider silk was completely degraded after 8weeks, hence painful changes or removing of wound dressings can be omitted. Furthermore, recent findings of an antimicrobial activity of native spider silk support the use as an antimicrobial active wound dressing [43]. Further research might use wound models colonized with bacteria to investigate advantageous properties of spider silk wound dressings for burn or chronic wound. Additionally, further studies should be taken out to confirm the suitability of spider silk and to prove that wound healing is really faster if wounds are covered with spider silk dressings. Taken together, we believe that spider silk might represent an interesting innovative biological wound dressing due to our promising results that might help in the care of burn wounds. Despite the limitations of the study mentioned above, this study implicates further investigations for the application of spider silk in wound healing as it appeared to be safe in use. It might display an interesting alternative to common wound dressings as it displays a biodegradable scaffold that favors ingrowth of keratinocytes and fibroblasts as well as capillaries without the need of removal.

Acknowledgments This article is dedicated to Kerstin Reimers. The in vivo-study and the CAM assay were funded by the Tui-Stiftung, immunoassay was funded by Else Kröner-Fresenius-Stiftung, VHX-5000 Live video microscope was kindly borrowed by Keyence. The authors are thankful to Kevin Hake for excellent support of the spiders as well as Anja Hillmer and Hanna Wendt for conducting live video microscopy and help with the CAM model.

Author contributions CL, FK and JWK performed the immunoassay, CL, FK, DV, SS and JWK performed histological analysis and took part in performing of live video microscopy, CL, SS and JWK helped with the CAM assay, CL, BM, KHW, PMV and JWK performed the operations of the sheep, VB and DV performed the PCR, CL, BM, KHW, SS and JWK did the conception and design of the study, CL, VB, DV, PMV, SS and JWK analyzed and interpreted the data. All authors took part in drafting and revising the article.

Disclosure There is no conflict of interest. All authors have read and approved the final article.

Please cite this article in press as: C. Liebsch, et al., Preliminary investigations of spider silk in wounds in vivo — Implications for an innovative wound dressing, Burns (2018), https://doi.org/10.1016/j.burns.2018.03.016

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Please cite this article in press as: C. Liebsch, et al., Preliminary investigations of spider silk in wounds in vivo — Implications for an innovative wound dressing, Burns (2018), https://doi.org/10.1016/j.burns.2018.03.016

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Please cite this article in press as: C. Liebsch, et al., Preliminary investigations of spider silk in wounds in vivo — Implications for an innovative wound dressing, Burns (2018), https://doi.org/10.1016/j.burns.2018.03.016