Silkworm silk scaffolds functionalized with recombinant spider silk

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Tissue Engineering and Regenerative Medicine

Silkworm silk scaffolds functionalized with recombinant spider silk containing a fibronectin motif promotes healing of full-thickness burn wounds Dimple Chouhan, Tshewuzo-u Lohe, Naresh Thatikonda, V GM Naidu, My Hedhammar, and Biman B. Mandal ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00887 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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ACS Biomaterials Science & Engineering

Silkworm silk scaffolds functionalized with recombinant spider silk containing a fibronectin motif promotes healing of full-thickness burn wounds Dimple Chouhan1, Tshewuzo-u Lohe3, Naresh Thatikonda4, VGM Naidu3, My Hedhammar4, *, Biman B. Mandal1,2 * 1Biomaterial

and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India. 2Centre

for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam,

India 3Department

of Pharmacology & Toxicology, National Institute of Pharmaceutical Education and Research, Guwahati, Guwahati 781032, Assam, India. 4Department

of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Center, Stockholm 106 91, Sweden.

*Corresponding authors: Dr. Biman B. Mandal Associate Professor Department of Biosciences & Bioengineering Indian Institute of Technology Guwahati - 781039, Assam, India Phone: +91-361-258 2225 Fax: +91 361 258 2249 (O) E-mail: [email protected] and [email protected] Dr. My Hedhammar Associate Professor Department of Protein Science School of Engineering Sciences in Chemistry, Biotechnology and Health KTH Royal Institute of Technology, AlbaNova University Center 106 91 Stockholm, Sweden E-mail: [email protected]

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Abstract Full-thickness cutaneous wounds, such as deep burns are complex wounds that often require surgical interventions. Herein, we show efficacy of acellular grafts that can be available off-the-shelf at an affordable cost using silk biomaterials. Silkworm silk fibroin (SF), being a costeffective and natural biopolymer, provides essential features required for the fabrication of 3D constructs for wound healing applications. We report treatment of third degree burn wounds using freeze-dried microporous scaffold of Antheraea assama SF (AaSF) functionalized with a recombinant spider silk fusion protein FN-4RepCT (FN-4RC) that holds fibronectin cell binding motif. In order to examine the healing efficiency of functionalized silk scaffolds, an in vivo burn rat model was used, and the scaffolds were implanted by a one-step grafting procedure. The aim of our work is to investigate the efficacy of the developed acellular silk grafts for treating fullthickness wounds as well as to examine the effect of recombinant spider silk coatings on the healing outcomes. Following 14‐day treatment, AaSF scaffolds coated with FN-4RC demonstrated accelerated wound healing when compared to the uncoated counterpart, commercially used Duoderm dressing patch and untreated wounds. Histological assessments of wounds over time further confirmed that functionalized silk scaffolds promoted wound healing, showing vascularization and re-epithelialization at an initial phase. In addition, higher extent of tissue remodelling was affirmed by the gene expression study of collagen type I and type III, indicating advanced stage of healing by the silk treatments. Thus, the present study validates the potential of scaffolds of combined silkworm silk and FN-4RC for skin regeneration.

Keywords: Silk scaffold; recombinant spider silk; burn wounds; wound healing; skin regeneration.


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Introduction Skin, the largest organ of our body plays multiple roles in protecting the body against external environment, pathogens and mechanical disturbances.1 Damage to the skin due to fullthickness wounds results in fatal consequences, leading to significant imbalance in physiological activities of internal organs. Cutaneous wounds are considered as a major healthcare problem due to an increasing number of trauma and burn cases.2 These are broadly categorized as full-thickness, partial-thickness and superficial wounds depending on the depth of tissue injury.3 The fullthickness wounds, often caused by thermal trauma or burns, impose great threat because such wounds lose the ability of self-repair and may lead to substantial organ failure in the absence of surgical interventions.2, 3 Burn injuries affect more than 11 million people every year, leaving majority of patients with disability, distress and discomfort.4-6 Burn injuries have contributed to almost 180,000 deaths every year according to the latest fact sheet by world health organization (WHO).7 The annual expenses on wound care may go beyond $ 22.4 billion by 2024, which was $ 14 billion in 2015.4 The golden standard approach, to apply autologous split-thickness skin grafts (STSGs), is dependent on the availability of patient’s healthy skin and faces a major drawback of secondary donor-site wounds.8 Apart from STSGs, allografts and cellular grafts (such as Apligraf and Dermagraft) containing allogenic cells have demonstrated significant improvement in the clinical outcomes.9,10 However, these approaches are associated with serious limitations such as high price, donor pathogens infection, short shelf-life, and two-step surgical procedures, which often requires an additional autologous split skin coverage.11, 12 Therefore, dermal reconstruction via acellular artificial grafts, which are available off-the-shelf, are desirable in case of emergencies or where patients have lost significant amount of skin tissue.



In this context, numerous natural biomaterials have been explored to develop acellular matrices, e.g. Integra based on bovine collagen and AlloDerm based on cadaveric skin. However, the high-cost of these products is still a major lacuna.13-15 This has led to an increasing research on exploring other natural biomaterials such as silk fibroin (SF) to improve the current situation of the healthcare system. Properties of SF such as biocompatibility, biodegradability, low immunogenicity and tissue-regeneration properties in the form of silk sutures have led to its increasing usage in the tissue engineering applications.16-18 Silk based constructs have shown quite a great progress in the field of tissue regeneration therapeutics in recent decades.18, 19 A recent study also proved that SF holds inherent property of wound healing via NF-ĸB signalling pathways, thereby having an impact on cellular recruitment during the healing process.20 The study demonstrated that SF helps in healing promotion by regulating the expression of proteins responsible for cell migration and proliferation such as vimentin, fibronectin and cyclin D1.20 Silkworm silk is widely available at low-cost and can be easily produced in large quantities through sericulture industries.16, 17 In India, a variety of silkworms are found, such as mulberry silk (Bombyx mori) and non-mulberry silk (Antheraea assama, Philsamia ricini and Antheraea mylitta).17 Among all silk types, B. mori silk fibroin (BmSF) and A. assama silk fibroin (AaSF) are well-studied in terms of their amino acid sequence, bioactivity and interaction with other materials.21-23 Both the silk variants have been well-explored in engineering of artificial tissues such as bone24, cartilage25, intervertebral disc26, liver27, blood vessels28, pancreas29 and wound dressing applications.30, 31, 32 We have previously reported studies of the interaction of BmSF and AaSF proteins with a recombinant spider silk protein, 4RepCT (4RC).22 The study revealed higher interaction of 4RepCT proteins with AaSF, owing to the possible similarity in their amino acid sequences.22 4RC is derived from the dragline silk of the Euprosthenops australis spiders, and


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produced via an Escherichia coli bacterial system by applying the recombinant DNA technology.33,

34

Taking advantage of the inherent interaction between the two silk types, we

successfully developed silk based wound dressings and bioactive scaffolds for skin regeneration applications.32 Herein, we aim to examine the healing properties of AaSF scaffolds coated with FN-4RC spider silk protein to treat full-thickness cutaneous wounds. The bioactive silk scaffolds are fabricated using the base material of AaSF sponges, which were subsequently top-coated with FN4RC, a recombinant spider silk fusion containing a cell binding motif derived from fibronectin (FN).35 Our target is to investigate the healing efficacy of the functionalized silk scaffolds towards wound repair and regeneration. The porous AaSF scaffolds produced by freeze-dried method have interconnected pores with an average pore size of 70 ± 20 µm, as determined in our previous study.22 The facile fabrication process of generating bioactive scaffolds through silk-silk interactions between silkworm silk bulk material and recombinant spider silk proteins has been well-established.22 The spontaneous self-assembly of 4RC proteins on the silkworm SF scaffold yields a fibrillar top coating of spider silk through physical adsorption, which enabled easy construction of stable bioactive silk matrices without any additional crosslinker.22 Herein, AaSF was preferred over BmSF bulk material due to comparatively higher interactions between AaSF and FN-4RC proteins, as inspected by the quartz crystal microbalance with dissipation assay.22 Our motivation behind applying the acellular FN-4RC coated silk scaffolds was to provide an artificial bioactive microenvironment to the platform that temporarily covers the wound-site and aid in rapid healing. To examine the healing efficacy of FN-4RC coated silk scaffolds under in vivo conditions, we used a third degree burn rat model and compared the results with non-coated silk scaffolds, a commercial wound dressing and untreated wounds. Both types of silk scaffolds



(coated/non-coated) were implanted on wound bed of debrided burn wounds in rats as temporary dermal substitutes via one-step grafting. We followed the stages of wound healing through histological study at various time-points. Assessment of angiogenesis, re-epithelialization and collagen deposition were performed through immunohistochemical staining to examine the key events of the healing process. The results of this study might provide insights into the wound healing process aided by the platform of bioactive silk scaffolds and interaction between host cells and silk materials.

Materials and methods Preparation of Antheraea assama silk fibroin (AaSF) solution A. assama silkworms (5th instar larva), procured from the sericulture farms of Assam, India were dissected and silk glands were separated from the larva. Silk fibroin was extracted out from the glands by carefully squeezing them using forceps following an established protocol.30 The isolated silk protein was washed multiple times with sterile water prior to dissolving in 1 % (w/v) sodium dodecyl sulfate (SDS) (Himedia, India) solution. The silk-SDS solution was kept at 4 °C for an hour and subsequently centrifuged at 5000 rpm for 5 min to remove the undissolved part. The soluble silk solution from supernatant was further dialyzed against water for 4-5 h under 4 °C conditions using 12 kDa MWCO dialysis membrane (Sigma-Aldrich, USA). Water was changed at a frequency of 1 h and the aqueous AaSF solution was collected. Fabrication of AaSF microporous scaffolds Microporous silk scaffolds were fabricated using 2.5 % (w/v) concentration of AaSF solution by freeze drying technique.32 AaSF solution (300 μL) was taken per well in 24-well plate


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and kept frozen at - 20 °C for 12 h. Lyophilization was carried out for 24 h to generate freeze-dried AaSF scaffolds, which were then treated with ethanol for β-sheet induction. Subsequently, the scaffolds were treated with sterile water multiple times to remove residual ethanol and autoclaved in wet condition for sterilization. Functionalization of AaSF scaffolds using recombinant spider silk proteins The AaSF scaffolds thus fabricated were coated with spider silk fusion protein by dip coating method.22 FN-4RC recombinant protein procured from Spiber Technologies AB, Sweden with the sequence TGRGDSPA from fibronectin fused to 4RepCT was used for coating purpose. The FN-4RC was diluted to 0.1 mg/mL concentration in 20 mM Tris buffer (pH 8.0) prior to coating the scaffolds. The AaSF scaffolds were incubated in the FN-4RC diluted solution for 1 h at room temperature for coating purpose. The excessive solution was subsequently removed, and scaffolds were washed with Tris buffer. The structure and morphology of AaSF scaffolds before and after coating of FN-4RC protein was observed by field emission scanning electron microscopic (FESEM) images (Figure S1, supporting information). The coated scaffolds were stored at 4 °C temperature conditions in wet state prior to grafting procedure. In vivo study design in burn injury model and treatment with microporous scaffolds The animal experiments on third degree burn injury model were performed according to the ethical approval received from ‘Institutional Animal Ethics Committee (IAEC)’, Guwahati medical college and hospital (GMCH), Guwahati, Assam, India (CPCSEA under the “Principles of laboratory animal care approval no. MC/916/2017/4”, Registration No. 351, 3/1/2001). Female wistar rats were procured from National Institute of Nutrition (NIN), Hyderabad, India weighing 200-250 grams each and were 6-7 weeks old. To create third degree burn model, standard protocol was followed on the rats and animals were acclimatized for 7-10 days in the animal house prior to



experiment. Anesthesia condition was given to the animals by administering K-X cocktail (Ketamine - 80 mg/kg and Xyalazine - 12 mg/kg) via intraperitonial route. Third degree burn to the rat skin was inflicted using a customized instrument to avoid injury to underneath organs and surrounding tissue following an established method.36, 37 An instrument consisting of soldering iron fitted with a cylindrical aluminum head of 1 cm diameter was used. A variable autotransformer was connected to the soldering iron to achieve precise control on the temperature of aluminum head. On setting the output of autotransformer to 120 V, necessary temperature of 80 °C was set which was measured by a Type K temperature probe attached to a digital thermometer. Third degree burn wound was inflicted on each rat by placing head of the device at 90° angle to the skin for 15 sec. The skin was folded parallel to the supporting platform to avoid any thermal insult to the deep tissues. Burn infliction was confirmed by the histological analysis of the necrotic burn injured skin in comparison to the normal skin (Figure S1, supporting information). Three groups of animals were taken considering 3 time-points (day 7, 14 and 21). Each rat was given only one wound and treatment groups were as follows: 1). Uncoated silk scaffold, 2) AaSF-FN (silk scaffold with a coating of FN-4RC), 3) Commercially available Duoderm (DD) wound dressing patch taken as positive control group and 4) Untreated group (UNT) taken as negative control group. Each time-point group consisted of four animals (n = 4). The Duoderm dressing patch (with specifications as ConvaTec Duoderm extra thin CGF dressing) was purchased from Amazon online store. Grafting of scaffolds was performed following clinical practices and established protocol.36, 38 A biopsy punch (1 cm diameter) was used to excise out the burn tissue after 48 h of burn infliction. Wounds were washed with sterile saline following debridement and scaffolds of wound-specific size were applied. To ensure attachment of scaffolds to the wound site, three skin staples were applied, which were subsequently removed after 3 days of application. The wounds


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with various treatment were also covered with an occlusive and transparent Tegaderm dressing for protection from infection and prevention from drying. Total three doses of antibiotic Ceftriaxone (20 mg/Kg) along with analgesic Meloxicam (5 mg/Kg) were given to all the animals via intramuscular route on every alternate day till day 7. In addition, the top dressings of Tegaderm was changed and grafted wounds were cleaned after every 2-3 days till 10-11 days. All the animals were placed in separate cages and monitored daily. Wound healing estimation Wound healing efficacy of different treatments was determined by calculating the wound size with respect to time. Photographs of wounds taken on day 0, 7, 11, 14 and 21 were analyzed to measure the diameter of wounds using Image J software. Wound healing efficacy was quantified by calculating the wound area with respect to original wound area using the formula – Wound area (%) = At/A × 100 Where At is the wound area at time (t) and A is the area of wound created at the very initial timepoint. Histological study Histological investigation of skin tissues was performed using Hematoxylin and Eosin staining (H & E) as per the manufacturer’s protocol. Images were taken under bright field microscope (EVOS FL, Life technologies, USA). For immunostaining, the sections were rehydrated and permeabilized by incubating in 0.1 % Triton X-100 solution for 10 min. Subsequently, 1 % bovine serum albumin (BSA) was used for blocking. Primary antibody antivWF (Abcam, UK) was then applied to the sections and incubated at 37 °C for 1 h. Herein, the sections of the tissues collected on day 7 were used to determine angiogenesis using von



Willebrand factor (vWF) marker. The sections were gently washed and subsequently stained using a secondary antibody conjugated with fluorescent molecule. Goat anti-rat IgG conjugated with Phycoerythrin (Sigma-Aldrich, USA) was applied and incubated for 1 h. Next, counter staining was done by incubating the stained sections in Hoechst 33342 (Invitrogen, USA) for 10 – 15 min, which stains nuclei. Finally, the sections were mounted and images were captured using a fluorescent microscope (EVOS FL, Life technologies, USA) to obtain overlapped images. Number of blood capillaries present in the stained sections was counted in 5 locations per section to obtain an average blood vessel density per section area. For immunohistochemistry (IHC) assay, Vectastain Universal ABC kit purchased from Vectors lab, U.K. was used according to manufacturer’s protocol. Primary antibodies, cytokeratin 10 (CK 10; 1 µg/mL), cytokeratin 14 (CK 14; 2 µg/mL), collagen type I (Col I; 5 µg/mL) and collagen type III (Col III; 5 µg/mL) were used. The primary antibodies were applied to various sections by incubating them in moisturized environment at 37 °C for 1 h. Further, the sections were washed and biotinylated secondary antibody was applied for 30 min. Next, the sections were incubated with ABC reagent for 30 min provided in the kit and subsequently 3,3′diaminobenzidine (DAB) reagent was applied to develop reaction product, which is brown in colour. Further, counterstaining of the sections was done with hematoxylin prior to mounting and images were captured. qRT-PCR analysis Gene expression study was performed using the skin tissues of specific time points (day 7, 14 and 21). The tissues stored in RNA Later solution brought to room temperature prior to their homogenization in TRIzol reagent (Sigma‐Aldrich, USA). Next, the mRNA extraction was performed by centrifugation of the samples at 13,000 rpm maintaining 4 °C to obtain the


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supernatant containing mRNA. Subsequently, chloroform was added to the collected supernatant in fresh vials and again subjected to centrifugation at similar parameters. The upper aqueous phase contained mRNA, which was further treated with isopropanol to obtain pure pellet of mRNA after centrifugation. The mRNA pellet thus isolated was dissolved in water (RNAase free water, Sigma‐Aldrich, USA). Subsequently, concentration of the obtained mRNA was measured using nanophotometer (Implen) and complementary DNA (cDNA) was produced through reverse transcription kit by PCR equipment (Verity, Applied Biosystems) according to the manufacturer’s instructions. The prepared cDNA was used to perform real-time PCR reactions using SYBR-Green PCR Mastermix procured from Applied Biosystems, USA. The expression of the gene of interest, namely, collagen type I, collagen type III and glyceraldehyde‐3‐phosphate‐dehydrogenase (GAPDH) was analyzed using primers listed in table S1 (supporting information). The average threshold cycle (Ct) values of the gene of interest (GOI) and the housekeeping gene (GAPDH) were obtained from the real time PCR reactions. Next, fold change of the GOI transcript levels in the particular sample group was compared with control group i.e. untreated sample group through the standard formula.39 Fold change = 2-ΔΔCt, where ΔΔCt = ΔCt(sample group) - ΔCt(control group) and ΔCt = Ct(GOI) Ct(GAPDH). Statistical analysis The experiments were performed considering n = 4 animals for each time-point group. Quantitative data (mean ± standard deviation) were calculated by Microsoft Excel and plotted using Origin 9.0 (Origin lab Corporation, USA) software. Statistical analysis of the data was performed through Origin software at the significant (** p ≤ 0.01) level by one-way analysis of variance (ANOVA) followed by the Tukey’s test.



Results and Discussion Rationale of the study Our simple approach of functionalizing silkworm silk scaffolds using bioactive spider silk fused with fibronectin domain is attributed to the inherent self-assembly property of spider silk over the silkworm silk matrices.22 Functionalization of silk has previously been done in other studies as well. For instance, silk fibroin protein containing additional RGD motifs was produced by transgenic silkworms for wound healing applications.40 The complex wound healing process require a platform over the wounds, which can be made bioactive by various strategies to guide the host cells.41 In another study, spider silk biomaterial functionalized with fibronectin type II domain was produced.42 However, production of high amount of such recombinant materials to be used as a bulk matrix increase the production cost by many folds. Herein, we aim to produce costeffective matrices by using inexpensive SF as bulk material, and functionalize it with a top-coating of FN-4RC spider silk containing RGD motifs (Figure 1a). The FN-4RC variant has been largely explored in the culture of variety of cells like fibroblasts, keratinocytes, endothelial, and stem cells.43,

44

The functional motif from fibronectin, including the RGD tripeptide, holds affinity

towards a broad spectrum of integrins present in various types of mammalian cells, which leads to improved cell attachment.43, 44 Cell adhesion is considered as an important factor for accelerating the healing process because it has roles in improved cell migration, growth and proliferation.1, 45, 46

Reinforcing silk scaffolds with enhanced cell binding activity using FN-4RC through our facile

fabrication process has enabled easy functionalization of matrices made up of silkworm silk. Applicability of such functionalized silk matrices was tested for the development of functional skin graft in our previous in vitro study.32 The bioactive silk scaffolds thus have the


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potential to treat difficult to heal wounds like third-degree burns. The coating procedure not only offered functional advantage but also led to a cost-effective approach. The silkworm silk used as a bulk material, requires a very low concentration (0.01 %) of recombinant spider silk fusion protein. Our approach thus circumvented the large-scale production problem associated with low yield of recombinant spider silk. Herein, the choice of biomaterial, i.e. AaSF porous scaffold and FN-4RC spider silk fusion protein, was based on our previous in vitro studies, which demonstrated higher cell proliferation of human dermal fibroblasts and endothelial cells in co-culture conditions in the FN-4RC coated AaSF scaffolds.32 The coated silk scaffolds also showed development of stratified epidermal layer formed on the scaffolds when co-cultured with keratinocytes under airliquid interface conditions, signifying the role of FN-4RC coating in development of skin tissue under in vitro conditions.32 The originality of the present work lies in the validation of the functional aspects of the bioactive scaffolds under in vivo conditions. The acellular scaffolds coated with FN-4RC were implanted in rats to understand the response of host cells towards skin regeneration by the support of bioactive silk scaffolds. The response of fibroblast, keratinocytes and endothelial cells was observed through histological study of the regenerated skin tissue, to examine the effect of FN-4RC coating for promoting wound healing. The in vivo study was accomplished using a well-established rat burn model as illustrated in the schematic image (Figure 1b and 1c). In control groups, positive control was taken using a commercially available Duoderm dressing patch (DD) and negative control was considered the untreated wounds (UNT) that did not receive any treatment. In addition, the effect of spider silk coating on the AaSF scaffolds were investigated by comparing with that of uncoated silk scaffolds to unfold the beneficial properties of FN-4RC on burn wound healing for the first time. In case of small wounds, where the self-repair mechanism of skin or the normal healing process is active, a



fibrin clot is formed that acts as provisional platform.47 However, in case of deep and large burn wounds, the self-repair mechanism fails and requires surgical intervention to cover the wounds and stimulate the healing process.12 Wound healing or skin regeneration is a cascade of complex series of events that require a provisional platform or support in the form of a temporary or permanent graft to direct the host cells towards regeneration of the novel tissue.41, 47 In addition, management of burn wounds is often difficult due to an extreme level of tissue necrosis.48 The microporous architecture of silk scaffolds provide an optimal environment for maintaining a moist milieu due to the moisture retention properties of silk, thereby preventing tissue necrosis of burn wounds.36 By recapitulating the cell binding ability of fibronectin through the FN-4RC coating on the porous silk scaffolds, we aim to trigger the natural healing process of skin to treat severe wounds like third degree burns.

One-step grafting of acellular silk scaffolds Implantation of silk scaffolds was performed via a one-step grafting procedure after 48 h of tissue debridement as demonstrated in figure 1c. Both the types of scaffolds (pristine AaSF and with top-coating of FN-4RC) were grafted at the injury site using surgical skin staples. The staples helped in holding the scaffolds in correct position of wound cavity at the time of grafting. Once the scaffolds were integrated with the adjacent skin tissue, staples were no longer required and were carefully removed after 3 days post-implantation. The implantation procedure used in the present study was simple, and did not disturb the wound-bed, to minimize the pain. This procedure indeed helped in one-step grafting of the scaffolds, which were found to be firmly attached at the wound-site.


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Figure 1: Schematic representation shows: (a) Image of the silk scaffold functionalized with FN4RC recombinant spider silk. (b) Illustration of the third degree burn wound model in rat system obtained using a customized metallic heating block. (c) Experimental set-up depicting treatment of cutaneous wounds in the burn rat model using silk scaffolds via one-step grafting procedure. The silk scaffolds were grafted using skin staples on the excised wounds post 48 h of burn infliction, which were removed on 3rd day. The animals assigned in each group of specific timepoints were terminated as marked by (T) for the examination of wounded tissue. In order to further validate the integration of scaffolds with wound-bed, the implanted graft site was excised out after 7 days post-implantation using a biopsy punch. Histological analysis of the implanted site clearly indicated integration of the implanted scaffolds with the host tissue as



visualized by an upper porous layer over the wound-bed (Figure 2). As a reference, histological analysis of wound cavity post-debridement was also performed to reveal creation of full-thickness wounds. The remnants of adipose layer and muscle layer of panniculus carnosus, as observed in the magnified image of the wound cavity, validated the loss of full-thickness cutaneous layer in the wounded region (Figure 2a). In comparison to the wound cavity, the wounds grafted with acellular silk scaffolds demonstrated presence of newly developed tissue that filled the wound cavity within 7 days (Figure 2b and 2c). In addition, invasion of host cells in the porous scaffolds was evident as depicted in the magnified images. The neo-tissue was also found infiltrating in the micropores of silk scaffolds, depicting signs of tissue ingrowth in the scaffolds, thereby attesting successful implantation of the scaffolds. The design of microporous scaffold was chosen in the present study because the interconnected pores of silk scaffolds provide a favourable architecture to the invading cells to migrate within the construct.47, 49 In addition, the porous microstructures help in tissue ingrowth and development of neo-tissue, which was beneficial in filling the wound cavity.47, 50 Cell infiltration and tissue ingrowth observed in the silk scaffolds demonstrated that the bioactivity provided by FN-4RC coating helped in rapid cell recruitment at the wounded site, which ultimately led to the formation of neo-tissue underneath the platform of silk scaffold.


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Figure 2: Histological study of the full-length wound biopsies demonstrate full-thickness wound creation and successful grafting of scaffolds by H and E staining: (a) Wound cavity formed after the debridement of necrotic burn skin; the cavity shows removal of full-thickness burn skin after the biopsy. The magnified image shows visible adipose (A) and muscle layer of panniculus carnosus (PC). (b and c) The images depict histology of wounded tissues treated with silk scaffolds on day 7 post-grafting; an upper layer of scaffold (S) well-integrated with the neo-tissue is clearly visible. Granulation tissue (G) developed in the place of wound cavity is also clearly visible underneath the scaffold layer. The magnified images reveal high infiltration of host cells and tissue ingrowth in the implanted scaffolds. Images on the right side represent magnified view (scale bar = 200 µm) of the region of interest as highlighted in the yellow box of low magnified images of cross section of full-length wound biopsies (scale bar = 1000 µm).

Wound healing efficacy of AaSF microporous scaffolds coated with recombinant spider silk for treating third degree burn wounds The support of the AaSF bulk material in the form of microporous scaffold was helpful in covering the wounds and aiding tissue ingrowth at the wound site. Significant difference in the



wound healing rate could be detected at the different time-points as the scaffold-treated wounds demonstrated faster wound healing in comparison to the control groups (Figure 3a). Among all treatments, the AaSF-FN scaffolds gave significantly faster wound closure rate, showing only 10.5 ± 2.48 % remaining wound area on day 14 compared to 19.8 ± 2.18 % by the uncoated AaSF counterpart. In contrast, UNT and DD showed 42 ± 3.29 % and 30.3 ± 2.35 % remaining wound area respectively, depicting delayed wound healing as observed on day 14. This indicated ≥ 2 fold accelerated healing rate by AaSF-FN scaffold treatment in comparison to control groups. Notably, early difference in the wound healing profile could not be observed due to presence of intact scaffold over wounds; however, improved healing was clearly evident in the later phase of healing. On day 14, significant differences in the healing profile could be observed for various treatments, depicting faster healing effects of both pristine uncoated AaSF and functionalized AaSF-FN scaffolds. The wound images also showed accelerated wound healing efficacy owing to the bioactivity of spider silk fusion protein present in the AaSF-FN scaffolds (Figure 3b). On day 21, AaSF-FN completely healed the wounds, showing no remnants of tissue eschar or scab. Nevertheless, significant difference was not observed between pristine AaSF scaffold and DD dressing on day 21, as remaining wound eschar could be observed on the wounds. On the other hand, UNT wounds showed relatively slower healing rate and demonstrated significantly higher remaining wound area on day 21 in comparison to all treatments. The gross morphology of implanted scaffolds also revealed firm attachment of scaffolds even after removal of staples till day 10. The results can also be validated by the histological examination of implanted scaffolds as shown in figure 2. This signified that once implanted at the wound site, the scaffolds promoted rapid tissue ingrowth that helped in adherence of the scaffold to the newly developed tissue matrix


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of wounds. We observed that the scaffolds gradually converted into a thin dry layer over the wounds and were then automatically removed after 10 days post-implantation. Representative images of the same have been highlighted in the inset, depicting morphology of dried scaffolds on day 10 and appearance of newly formed skin underneath the scaffold (Figure 3c). Auto-removal of scaffolds post 10 days of treatment might be attributed to the development of neo-dermal matrix formed beneath the scaffold, which pushed the outer scaffold layer after tissue maturation. Another reason might be the higher mechanical strength and slow biodegradation rate of SF scaffold, which did not contract and remodel along with the wound contraction process. Instead, the scaffolds acted as a provisional matrix and supported development of neo-tissue beneath. Higher wound contraction soon after scaffold removal demonstrated that the scaffolds stimulated cells for faster healing and promoted tissue ingrowth at a faster rate. In addition, removal of scaffolds in the later phase of healing process did not lead to any major disturbance in the wound matrix since wound healing cascade was already stimulated by the scaffolds in the earlier phase.



Figure 3: Wound healing examination of various treatment shows accelerated healing efficacy of AaSF-FN scaffolds containing coating of bioactive spider silk fusion protein as determined by (a) The wound area measurement at various time-points depicting significantly faster wound closure by the treatment of scaffolds coated with FN-4RC spider silk fusion protein, ** represents p ≤ 0.01. (b) Pictures of the wounds at various time-points during the wound healing process, scale bar = 10 mm. (c) The inset shows morphology of scaffolds over the wounds and development of regenerated skin visible after removal of scaffolds on day 10.

Histological examination of burn wounds during the treatment Histological analysis of the tissue specimens collected on day 7, 14 and 21 revealed timely healing response by the grafted scaffolds (Figure 4). Granulation tissue assessment on day 7 indicated early healing response in case of treatment with AaSF scaffolds, as seen by the presence of neotissue ingrowth underneath the scaffold layer (Figure 4a). On closely observing the tissue


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section of wound matrix treated by pristine AaSF scaffold, gaps were found in the middle portion of the neomatrix (highlighted by dotted yellow line). Such evident gaps were not found in the wounds treated with AaSF-FN, suggesting more recruitment of cells leading to uniform distribution of mature granulation tissue at an early stage of the healing process. The coating of FN-4RC on top of AaSF scaffold clearly demonstrated better wound healing efficiency in comparison to uncoated scaffolds. Fibronectin present in the natural fibrin clot is the most studied protein in wound healing applications, as it plays major role is recruiting cells owing to the presence of a cell adhesion motif (RGD tripeptide).46, 51 The RGD sequence present in fibronectin binds to a broad range of integrins present on cells and is highly explored in cell binding mechanism.43, 44 The cell binding mechanism plays a major role in healing the wounds due to enhanced cell migration and proliferation.45-47, 51 Thus, AaSF-FN scaffolds acted as a bioactive platform over the wounds and could stimulate the healing at a faster rate. Among the control groups, treatment with DD dressing demonstrated development of granulation tissue equivalent to treatment with the pristine AaSF scaffold. However, the UNT group showed delayed wound healing response, characterized by a very thin tissue layer over the adipose layer at the wound site. This suggested that in contrast to the treated wounds, granulation tissue was not developed in the untreated wounds till day 7 post-operation. On day 14, the arrangement of neotissue appeared well-organized, suggesting progression in the wound healing in the treated wounds. Untreated wounds showed granulation tissue development on day 14, which was not formed on day 7, confirming delayed healing process in the untreated wounds. Among the treated groups, AaSF-FN scaffolds demonstrated a growing thick epidermal layer on the uppermost surface of neomatrix signifying early onset of reepithelialization (Figure 4b). In contrast, neither wounds treated with pristine AaSF nor DD



dressing did show any thick epidermal layer. However, complete re-epithelialization was observed on day 21 in all the treated groups (Figure 4c). Except for the UNT group, all the treated wounds demonstrated presence of a thick mature epidermal layer. Absence of epidermal layer in the UNT group further corroborated delayed healing profile of untreated wounds as also observed in the wound images seen in figure 3b. AaSF-FN showed well-formed dermal and epidermal structures as confirmed by the higher magnified images of tissue sections, suggesting improved healing efficacy owing to presence of the FN-4RC spider silk protein. The additional top-coating of bioactive spider silk on scaffolds aided in guiding the cells towards accelerated healing and tissue development owing to its cell conducive microenvironment. Similar results were also obtained in our previous study of diabetic wound healing, which demonstrated improvement in angiogenesis and re-epithelialization of wounds by the treatment of FN-4RC coated nanofibrous mats.52


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Figure 4: Hematoxylin and eosin stained tissue sections of the regenerated skin biopsies taken at various time-points depict healing progress by different treatment: (a) development of granulation tissue at an initial phase by the silk treatment as shown by the neo-tissue formed at the wounded site on day 7, (b) beginning of the re-epithelialization event in the AaSF-FN treated wounds, depicting the role of FN-4RC coating in accelerating the development of epithelial layer by day 14 and (c) dermo-epidermal development in the wounds treated with silk scaffolds as represented by the mature epidermal and dermal layers of the regenerated skin at the wounded site on day 21. The low magnified images (4 x magnification) demonstrate histomorphology of the cross section of tissue (scale bar = 1000 µm); the high magnified images (20 x magnification) represent morphology of the selected portion (scale bar = 200 µm) as highlighted in black box.



Angiogenic potential Angiogenesis is one of the vital determinants of wound healing progression that indicates proliferation of connective tissue at the initial stage of the healing process.53 Therefore, we evaluated the angiogenic potential of various treatments by immunofluorescence using vWF marker of endothelial cells (Figure 5a). Among all treatments, the wounds treated with AaSF-FN scaffolds demonstrated significantly higher vessel density and higher number of mature blood capillaries in the granulation tissue, indicating signs of angiogenesis and higher recruitment of endothelial cells. The results were consistent with the superior regenerative response, also observed in the H & E stained sections (Figure 4a). The newly formed blood vessels during the initial wound healing phase help in the supply of oxygen and nutrients to the newly developed granulation tissue. Coating of FN-4RC on the scaffolds provided a local microenvironment suitable for cell adhesion and thereby resulted in higher angiogenesis in the AaSF-FN treated wounds. Formation of a few blood vessels were observed in wounds treated with both pristine AaSF scaffold and DD dressing, depicting presence of growing granulation tissue. However, mature blood capillaries were not observed in the UNT group, as only few endothelial cells could be visible in scattered fashion. Neo-vascularization, as observed by the blood capillaries in the granulation tissue, also confirmed angiogenic and healing potential of silk proteins. Quantification of blood vessels further validated the angiogenic potential of FN-4RC coated scaffolds (Figure 5b). The wounds treated with AaSF-FN scaffolds demonstrated higher vessel density when compared to the uncoated AaSF scaffold treatment and control groups, p ≤ 0.01. The study also indicated that the coating of FN-4RC spider silk fusion protein on the AaSF scaffolds improved the angiogenesis at an early stage of wound healing process.


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Figure 5: (a) Examination of the extent of angiogenesis in the skin biopsies collected on day 7 using anti-vWF immunostaining shown by the blood capillaries stained with red colour (vWF); the blue colour depicts nuclei in the tissue section stained with Hoechst 33342 (Scale bar = 200 μm). (b) Graphical representation of the vessel density by quantifying the number of blood vessels, depicting significantly higher vessel density in the wounds treated with silk scaffolds having spider silk coating (AaSF-FN) in comparison to that of other groups, ** represents p ≤ 0.01.

Re-epithelialization assessment We further investigated the re-epithelialization event of wound healing process by immunohistology to examine the development of epidermal layer over the wounds by different treatments. We investigated the chief markers of epidermal layer, namely, CK 10 and CK 14, in the tissue sections collected on day 14 and day 21 through IHC assay (Figure 6). On day 14, CK 10 expression was not observed in the uppermost layer of wound matrix in any of the groups due to absence of mature epithelial layer within this period (Figure 6a). However, wounds treated with AaSF-FN demonstrated expression of CK 14 in the growing epithelial tongue on day 14, as highlighted by a black arrow in the respective image (Figure 6b). The same section did not reveal expression of CK 10. This support our hypothesis that the epidermal layer observed on the uppermost layer in wounds treated with AaSF-FN was immature and expressed only CK 14



marker. On day 21, CK 10 expression was manifested in the suprabasal layers of the epidermal region of wounds treated with AaSF and AaSF-FN scaffolds, indicating the positive role of silk protein in re-epithelialization. Untreated wounds and those treated with DD did not demonstrate expression of CK 10 even on day 21, revealing immature epidermal layer still in the growing stage due to delayed healing. CK 14 expression was observed throughout the epidermal layer in all the treated wounds, signifying re-epithelialization of the wounds within 21 days of treatment. Moreover, the IHC images also revealed well-formed epidermal structures (expressing both CK 10 and CK 14) in wounds treated with AaSF and AaSF-FN, indicating improved healing efficacy of silk biomaterials in comparison to the control groups. The coating of FN-4RC spider silk on silk scaffolds helped in early re-epithelialization of wounds and mature development of epidermal layer. Notably, the wounds treated with AaSF-FN scaffolds demonstrated a budding epithelial tongue over the neo-tissue as observed by the expression of CK 14 on day 14, thus revealing beginning of re-epithelialization in the mid-stage of wound healing. This indicated that FN-4RC played a major role in re-epithelialization of wounds. The coated silk scaffolds demonstrated enhanced cell-material interactions and formation of bilayer keratinized skin layer in the in vitro studies conducted in our previous work.32 The study highlighted keratinization potential of FN-4RC coating when cellular scaffolds were cultured under air-liquid interface conditions.32 By providing artificial microenvironments containing FN motifs through silk materials, we could achieve the same effect of improved re-epithelialization under in vivo conditions.


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Figure 6: Re-epithelialization assessment of wounds on day 14 and 21 by immunohistochemistry assay of the sections using cytokeratin markers (a) CK 10 and (b) CK 14, showing gradual development of the epidermal layer during the skin regeneration process. Tissue sections of wounds treated with AaSF-FN reveal development of growing epithelial tongue on day 14 with marked expression of CK 14 (highlighted by black arrow) showing early stage re-epithelialization. Brown colour depicts presence of particular cytokeratin marker and purple blue colour depicts nuclei stained with hematoxylin. (Scale bar = 100 μm).

Wound Remodelling Assessment: Gene Expression Study and Immunohistology Finally, we examined the effect of various treatments on the dermal remodeling by analyzing the expression and deposition of collagen type I and collagen type III fibers in the regenerated skin biopsies. The real time qPCR revealed significant remodeling between collagen



fibers in the wound matrix from day 7 to day 21. The expression of Col I on day 7 indicated early healing profile in case of wounds treated with AaSF and AaSF-FN scaffolds, as the expression was 3-4 fold higher (Figure 7a). In contrast, the commercial dressing showed only 1.5 fold increment in Col I expression, suggesting slower healing in comparison to wounds treated with scaffolds. Col I was found to be upregulated on day 14 and day 21, validating tissue formation in the later stages of healing process. In wounds treated with AaSF, the expression on Col I was highest on day 14, confirming ongoing healing stage of the wounds. On the other hand, significantly lower expression on day 21 demonstrated remodeling stage in the later phase of wound healing. Similar expression behavior was observed in the wounds treated with AaSF-FN. Higher expression of Col I on day 7 and day 14 indicated early granulation tissue development and tissue ingrowth in the wound cavity, suggesting accelerated wound healing profile. A significant regression in the Col I expression on day 21 compared to day 7 and 14 indicated a remodeling stage, as the healing process reached the final stage. The expression study of Col III also indicated similar healing pattern (Figure 7b). The expression continued to upregulate from day 7 to day 21 in case of DD treatment. This confirmed the ongoing healing phase of wounds throughout the time-period of 3 weeks. In contrast, wounds treated with silk scaffolds demonstrated initial upregulation and regression thereafter, indicating a remodeling stage. Expression of Col III was found to be significantly higher on day 14 in comparison to day 7 and day 21 in wounds treated with AaSF scaffold. This clearly showed that the highest secretion of collagen type III fibers was during the mid-stage of the healing process, in order to promote wound contraction. Once the wound contraction was completed, the expression was regressed on day 21 in the remodeling stage. The dermal layer of cutaneous system majorly consists of 80 – 85 % collagen type I fibers and 10 – 20 % collagen type III fibers, which


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collectively provide integral stability and tensile strength to the skin.54-56 The secretion of collagen type III remains more or less constant in the intact skin; however, the secretion increases in the wounded skin in order to facilitate wound contraction with the help of collagen type III fibers.56, 57

Therefore, early secretion of collagen type III fibers is also considered as a sign of wound

contraction and accelerated wound healing process.56 Further, in case of normal healing process, the deposition of collagen fibers diminishes with time because persistent secretion of collagen fibers is associated with scar or keloid formation.56, 58 Higher expression of collagen fibers in the mid-stage (day 14) and lower expression in the later phase of wound healing (day 21) by silk scaffold treatments indicated regulated expression of collagen fibers. Overall, the results suggest that wounds treated with silk scaffolds were at a more advanced stage of healing compared to control groups.

Figure 7: Fold change in the expression of (a) Collagen type I gene and (b) Collagen type III gene in the regenerated skin tissue collected at various time-points demonstrating remodeling of extracellular matrix during the complete course of healing process. Fold change signifies the expression of gene in the sample group with respect to the untreated wounds, **p ≤ 0.01.

IHC assay on the tissue section using antibodies against collagen type I and collagen type III proteins depicted morphology of collagen fibers deposited in the regenerated skin on day 21



(Figure 8). Mature bundles of collagen type I were visible in the dermal layer of wounds treated with silk scaffolds (Figure 8a). Such well-organized collagen bundles were not visible in the untreated wounds, which might be due to the incomplete healing of wounds by day 21. Further, examination of the collagen type III fibers through IHC showed interesting results in the dermal regions of the silk treated wounds (Figure 8b). Notably, wounds treated with AaSF scaffolds demonstrated collagen type III fibers on the edges and not in the center of wound matrix. This specified the typical behavior of collagen type III fibers in wound contraction from the edges towards the central wound matrix. Such morphological observations also revealed that the collagen remodeling was still in the incomplete phase by day 21 in wounds treated with AaSF. On the other hand, wounds treated with AaSF-FN demonstrated homogeneously distributed fibers of collagen type III all over the dermal regions, suggesting characteristics of mature skin regeneration. The results also verified modulation of provisional granulation tissue towards a more resistant and mature dermal matrix within 21 days, revealing final stage of the wound healing process.


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Figure 8: Immunohistochemistry analysis depicts (a) Collagen type I and (b) Collagen type III fibers in the healed skin tissue and their deposition on day 21. The low magnified images (10 x magnification) represent cross section area of the regenerated skin tissue (scale bar = 400 µm); selected portion as highlighted in black box marks the high magnified images (40 x magnification) of the region of interest (scale bar = 100 µm). Brown colour depicts collagen fiber and purple blue colour depicts nuclei stained with hematoxylin.

The platform of silk scaffolds guided in the regular deposition of collagen fibers during the healing process. The detailed study proved our hypothesis that the functionalized silk scaffolds helped in accelerating the natural healing pathways characterized by the granulation tissue



development at an initial healing phase (day 7), early beginning of re-epithelialization (day 14) and regeneration of dermal structures showing tissue remodelling by day 21.

Conclusion Combining our findings of the present study, one-step grafting of silk scaffolds offered a facile way to treat full-thickness wounds with an accelerated healing rate. Despite having the intrinsic regenerative properties, additional functionalization of silkworm silk with bioactive recombinant silk facilitated accelerated wound healing. With the help of recombinant functionalized bioactive spider silk fusion proteins, bulk material in the form of porous silk scaffold could be easily functionalized and could be applied in the form of acellular temporary grafts. The encouraging results by spider silk coated scaffolds reveal outstanding contribution of the combined biomaterials for wound healing applications. Thus, our simple strategy of constructing bioactive acellular scaffolds demonstrated great potential of possible treatments of burn wounds at an affordable cost. Future validation of the study may be performed in other animal wound models, which are close to human skin. It is evident from the present study that the functionalized silk scaffolds in the form of acellular grafts, demonstrated great potential in healing full-thickness cutaneous wounds. The silk scaffolds showed accelerated wound healing and temporary integration with the host tissue, thereby facilitating angiogenesis and early re-epithelialization of wounds. The silk scaffolds implanted through one-step grafting procedure provided a functional niche in the form of an artificial provisional matrix over the wounds. The bulk material of SF, being a cost-effective material may provide an additional advantage over the currently used highly-priced products. Overall, the present work demonstrated that the functionalized silk scaffolds can be used in the


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form of a ready-to-use, off-the-shelf acellular graft. The facile approach of using combined spider silk and silkworm silk biomaterials in treating difficult wounds like burn injuries encourages a conceivable translation from bench to bedside in future.

Supporting Information Table S1, primer sequence used in the gene expression study; Figure S1, FESEM images of silk scaffolds before and after coating of FN-4RC; Figure S2, histology of normal rat skin versus necrotic skin post burn injury.

Acknowledgements Spiber Technologies AB, Sweden is highly acknowledged for providing the recombinant spider silk protein FN-4RepCT. The project was funded through Department of Biotechnology (DBT; BT/IN/Sweden/38/BBM/2013), Government of India, together with Vinnova (Grant 201304641), Formas (Grant 221-2013-883), the Swedish Research Council (Grant 2013-10-25), and Knut and Alice Wallenbergs Stiftelse (Wallenberg Academy Fellows 2013). BBM also acknowledges the generous funding support through the Department of Biotechnology (DBT) and Department of Science and Technology (DST), Govt. of India under various funding programs. IIT Guwahati central instrumentation facility is acknowledged for use of high-end equipment.

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Silkworm silk scaffolds functionalized with recombinant spider silk containing a fibronectin motif promotes healing of full-thickness burn wounds Dimple Chouhan1, Tshewuzo-u Lohe3, Naresh Thatikonda4, VGM Naidu3, My Hedhammar4, *, Biman B. Mandal1,2 * 1Biomaterial

and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India. 2Centre

for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam,

India 3Department

of Pharmacology & Toxicology, National Institute of Pharmaceutical Education and Research, Guwahati, Guwahati 781032, Assam, India. 4Department

of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Center, Stockholm 106 91, Sweden.


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Silkworm silk scaffolds functionalized with recombinant spider silk

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