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Silk-Based Biomaterials in Cutaneous Wound Healing

A Systematic Review

Kamalathevan, Pragash MA (Cantab), MBBS (UK); Ooi, Peng S. MBChB (UK); Loo, Yew L. MBChB (UK)

doi: 10.1097/01.ASW.0000546233.35130.a9
FEATURES: LITERATURE REVIEW
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OBJECTIVE: Effective wound dressings should promote healing through cellular migration, neovascularization, and re-epithelialization. Silk fibroin (SF) and silk sericin (SS) are reported to have very good biocompatibility, excellent mechanical properties, and controlled biodegradability. This review investigates the use and performance of silk-based biomaterials in cutaneous wounds within in vitro, in vivo, and randomized controlled studies.

METHODS: Study authors conducted a comprehensive literature search on the use of silk-based dressings in cutaneous wound healing and investigated reports of the advantages and disadvantages of SF and SS along with these materials’ methods of characterization, cell migration, neovascularization, wound closure, and cytotoxicity.

RESULTS: In vitro and in vivo animal models have shown SF-based biomaterials promote good cellular adhesion and fibroblast proliferation in cutaneous wounds. The porosity and silk concentration of silk-based scaffolds are key determinants of biodegradation and plasmatic imbibition capabilities and can help promote wound healing. In reviewed studies, SF biomaterials promoted neovascularization as early as 7 days and better than common dressings, demonstrating low cytotoxicity and immunogenicity. That said, a concern with the use of SS is the tendency to cause a hypersensitivity reaction.

CONCLUSIONS: The benefits of silk-based biomaterials seem evident based on promising preclinical studies. Both SF and SS have been shown to have excellent wound healing properties by promoting cell attachment, migration, and collagen deposition. The authors encourage the use of SF and SS in more trials for wound healing.

In the Department of Surgery and Interventional Science at University College London in the United Kingdom, Pragash Kamalathevan, MA (Cantab), MBBS (UK); Peng S Ooi, MBChB (UK); and Yew L Loo, MBChB (UK), are Burns, Plastic & Reconstructive Surgery Candidates. The authors have disclosed no financial relationships related to this article. Submitted April 23, 2018; accepted in revised form June 22, 2018.

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INTRODUCTION

Recent research has examined nanofibrous matrices, hydrogels, and other natural or synthetic polymeric scaffolds including those derived from silk that are biocompatible and biodegradable, mimicking the skin’s extracellular matrix.1,2 Because of their porosity and excellent mechanical strength, as well as cellular and molecular modulators that stimulate tissue redevelopment, these scaffolds are well positioned to create a beneficial microenvironment for wound healing.3

The spun fiber that is silk consists of two proteins, a central protein known as a fibroin and a glue-like coating known as sericin. The arrangement of silk fibroin (SF) involves β-sheet crystal regions with hydrogen bonding as well as semicrystalline regions (Figure 1).4 Sericin has been reported to cause problems with silk biocompatibility.2,5 Sericin-free SF has been found to exhibit very good biocompatibility.4,6,7

Figure 1

Figure 1

This systematic review discusses the application of SF and silk sericin (SS) for treating burns and other cutaneous injuries, and their respective advantages and disadvantages and evidence based on in vitro, in vivo, and randomized controlled studies.

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METHODS

The authors of the present study conducted a comprehensive literature search using the electronic databases PubMed, EMBASE, and MEDLINE (Ovid) for studies on the advantages and disadvantages of SF and SS in wound dressings. Keywords included “silk fibroin” or “sericin” and “wound dressings” or “mechanical properties” or “biocompatibility” or “in vivo” or “in vitro” or “biomaterials” and “wound healing.“

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Data Extraction

Inclusion criteria for this study were original studies and reviews published in English between 1985 and 2017. In vivo, in vitro, and human studies were evaluated for reported advantages and disadvantages associated with the use of SF and SS in wound dressings. Studies were categorized as supporting evidence if they had original data from formal assessments and anecdotal opinions if they were mentions of use without any formal assessments or measurements.

Exclusion criteria were articles published in languages other than English, letters to the editor, discussions, and abstracts.

Because of the heterogeneity of each study design and outcome report, statistical analysis was not possible in this review.

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RESULTS

A total of 29 studies were identified. Fifteen discussed the advantages and disadvantages of SF as a biomaterial (Table 1), 1 discussed both SS and SF as a biomaterial, and 13 articles discussed SS only. Only one human study was identified, a randomized controlled trial. The rest were either in vitro or in vivo studies.

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Silk Fibroin

Biomaterials with SF were reported to have excellent mechanical properties.1,2,8,9 In Marsano et al’s9 study on blended regenerated cellulose-SF fibers, they found that a ratio of 75:25 had better mechanical properties with higher modulus compared with a pure cellulose control. Li et al,8 in their study on an SF/sodium alginate blend, reported similar results.

Song et al5 mentioned that SF blends have good biocompatibility but did not perform any formal investigation to support their statement. There were four articles that anecdotally described SF blended biomaterials as having controlled biodegradability1,2,5,8 and four that reported (also anecdotally) SF does not greatly induce immune response when used in vivo.2,5,10,11

Li et al8 reported that although SF with β-sheet form has better mechanical properties, random coil conformation increases hydrophilicity of SF-blended wound dressings. The water contact angles of their SF/sodium alginate–blended films were generally lower compared with their control film. However, water uptake capacity is also affected by the concentration of SF in the blend.12 Reduced reuptake ability was observed in a 50% SF blend1 because excess SF can affect the polymer surface structure.

Only one study reported the blood compatibility of SF-blend materials. Sakabe et al’s13 in vivo experiments using fibroin-coated sutures buried in animal connective tissue found that there was no evidence of thrombus formation at days 1 and 14 of their experiment. Li et al8 reported only supportive anecdotal opinions on the blood compatibility of SF biomaterials.

Further, SF-blended materials have high porosity that makes them ideal biomaterials.1,2,5,12,14,15 The porosity of SF materials can be assessed via scanning electron microscopy.1,5,8,12,16

Despite the many advantages of SF, it is not without disadvantages. Pure SF films in a dry state have very poor mechanical properties and cannot be used as a wound dressing.8 Electrospun SF nanofibers increase material porosity and can ironically provide a suitable environment for bacterial growth.5,17 The brittleness of regenerated SF causes it to fragment easily and makes it difficult to create a uniform thickness of the material.1,18 Silk also has no antimicrobial effect;8,19 many studies blend silk with other polymers with infused antimicrobial properties or antibiotics such as vancomycin21 and antimicrobial peptide.5

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Silk Sericin

Silk sericin is a natural polymer that joins two SFs in order to form silk yarn. Despite certain literature reporting on the poor biocompatibility of biomaterials containing SS, the molecule is highly hydrophilic and its structural composition, solubility, and organization enable cross-linking and combination with other polymers. Kunz et al20 anecdotally reported that SS has antioxidant, moisturizing, proproliferative, and cell attachment properties (Table 2). It has also been reported that SS increases cell proliferation.21

With regard to the increase in cell attachment, Tsubouchi et al’s22 study on human skin fibroblasts cultured on sericin-coated Petri dishes reported an increase in living cell number count after 72 hours of 250% when compared with control. To study the antioxidant activity of SS, Chlapanidas et al23 measured reactive oxygen species (ROS)–scavenging activity and antityrosinase activity using the 2,2-diphenyl-2-picrylhydrazyl hydrate method and antityrosinase activity assay, respectively. They found that SS significantly and positively influenced ROS-scavenging activity and antityrosinase activity (P < .0001).

One of the main disadvantages of using SS-based polymers is the tendency to induce a hypersensitivity reaction. Altman et al,10 in their review of materials (mainly sutures with virgin silk), found that SS caused a type I hypersensitivity reaction as well as a delayed hypersensitivity reaction because it upregulated immunoglobulin E (IgE), causing an asthmatic reaction in some patients. A prospective cohort study in India reported a high sensitivity to silk filature among workers.24 A skin prick test was used to determine silk allergen sensitization; of the 120 participants who were exposed to SS, 35 of them (29.17%) were sensitive to it.

In a case study by Wen et al,25 the same skin prick test was done in 64 children younger than 15 years. It was discovered that 75% of them had an upregulation of IgE as well as signs of a delayed hypersensitivity reaction such as allergic rhinitis and conjunctivitis. In an earlier study by Dewair et al,26 there was an upregulation of IgE after a skin patch test in human subjects; materials used were a silk rug and silk material that contained a high percentage of sericin. Sobajo et al’s4 review of silk-based biomaterials anecdotally stated that sericin caused an up-regulation of IgE in surgical patients as well as a type I hypersensitivity reaction.

Kaur and colleagues’17 study on Escherichia coli growth on agar plates containing both SF and SS found that there was an increase in bacterial concentration 103 colony-forming units/mL in the SS group compared with SF. All samples and equipment underwent autoclaving prior to the experiment to increase the accuracy of the results.

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DISCUSSION

Physical Properties and Material Characterization of Silk Polymer–Based Scaffolds

Material characterization of a product plays a crucial role in determining how the biomaterial performs as a medical device. In the literature, various techniques have been used to characterize the physical and chemical properties of silk polymer. Scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction, atomic force microscopy, and measuring water contact angles are commonly applied to study the secondary structure of silk-based scaffolds.

Scanning electron microscopy is widely used to study the external morphology and average pore size of these scaffolds and has shown well-structured and interconnected pores formed by random nanofilaments tangled with each other (Figures 2A, B).1 Sufficient pore size is required to allow the cells to move within the scaffold in the process of wound healing and regeneration;27 optimum pore size to support adult mammalian skin regeneration ranges from 20 to 125 μm.28 Most of the SF-based scaffolds in the literature were manufactured by electrospinning, which allows researchers to manufacture a scaffold with controllable pore size to allow cell migration and proliferation.

Figure 2

Figure 2

In the Fourier transform infrared spectroscopy spectra, SF and SS commonly present in three characteristic bands. They are amide I (1,700–1,600 cm-1), II (1,540–1,520 cm-1), and III (1,300–1,220 cm-1), which correlate with C=O stretching, N-H bond bending, and a combination of C-N stretching and N-H deformation, respectively.29 Amide I and II bands are strongly related to the β-sheet, whereas the amide III band is related to random coil formation.29

X-ray diffraction studies of SF scaffolds indicate the presence of an amorphous random coil matrix with a poorly orientated crystalline domain of β-sheet.29 Comparatively, SS has a higher content of random coils with some β-sheets, which makes it behave like an amorphous material.30–32 This makes sericin a fragile material on its own, which is accompanied by a broad molecular weight range and high water solubility.33 Fortunately, SS consists of polar side chains, which allows it to be cross-linked.33 Treatment with ethanol can also induce aggregation of the protein to transform random coils into β-sheets by fractionation or dehydration.30,32

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In Vivo and In Vitro Studies

The application of SF in tissue engineering and regenerative medicine is common because of its positive biologic and mechanical properties.10,34 Numerous in vitro and in vivo studies have been performed in recent years looking specifically into the cell viability, macrophage response,35–37 and vascularization capacity38 of SF to support its potential application as a wound dressing in the biomedical field.

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Cell Attachment, Migration, and Proliferation

In vitro studies have shown that nonwoven SF is biocompatible with human cells and can support the growth of a variety of human cell types including epithelial cells, fibroblasts, glial cells, keratinocytes, osteoblasts, and endothelial cells.10,27,39–41 Most epithelial cells can multiply and spread along SF fibers by day 3, eventually forming a tissue-like structure after filling the spaces available between the fibers.39 This supports the use of SF-based scaffolds to promote the adhesion and growth of human fibroblast cells.

Although engineered scaffolds should promote cell infiltration, they must not do so in an inappropriately prolonged manner. As much as the initial inflammatory phase is important to direct cell movement, a heightened or prolonged phase will prevent adequate long-term healing of the wound.42 Baoyong et al43 investigated the use of spider SF in deep second-degree burns within a rat model. Histologic examination showed moderate inflammation with macrophages from days 3 to 5 in both the silk group and a negative control group. However, the degree of leukocyte infiltration was suppressed in the SF group by day 7 with the development of de novo connective tissue. In the negative control, there was still increased inflammatory infiltrate up to day 14. Supporting studies have shown SF to limit the acute inflammatory phase to within 14 days after injury.44 Taken together, these studies show SF produces less inflammation and neutrophil/leukocyte infiltration compared with control and certain alternative dressings.45 Silk fibroin can therefore facilitate the acute leukocyte infiltration but not detrimentally prolong the inflammatory phase.

Similarly, studies with SS-based wound dressings have shown that these membranes promote only a moderate inflammatory response.46 Akturk et al46 further showed that glutaraldehyde–cross-linked sericin/collagen membranes also successfully biodegrade in 3 weeks; the biodegradability characteristic makes sericin an excellent wound dressing material.

Fibroblasts play a significant role in wound healing by promoting the deposition of collagen. Wound healing scratch assays have shown SS to facilitate the migration of fibroblast L929 cells and keratinocytes at levels as low as 100 μg/mL.47,48 Complementary in vivo studies give compelling evidence that SS provides good structural support for cell attachment and migration, resulting in collagen deposition/fibroblast proliferation and attachment, both hallmarks of wound healing.

Gil et al49 showed that SF dressings provided adequate cell adhesion and migration on wound sites compared with empty sites. Of note, Masson trichrome staining and histologic quantification revealed denser collagen regeneration within SF biomaterial–dressed wounds compared with both empty wounds and hydrocolloid-dressed wounds. Subsequent studies have shown that the morphology of deposited collagen is also largely different in silk biomaterial–dressed wounds.50 Collagen bundles in SF-treated wounds within a rabbit model were more stout and wavy, with moderate intercollagen gaps attributed to scarless healing.50

Roh et al51 further reinforced these findings. Collagen deposition of granulation tissue was significantly increased 7 days after inducing full-thickness wounds in rats treated with SF compared with control (untreated wound defects; P < .05). These studies support the use of SF as a structural scaffold because it not only increases the deposition of extracellular matrix content but does so in the correct orientation. Both of these features are imperative for adequate wound healing while also promoting good aesthetic results.

Similar studies have been done with SS-based biomaterials in the context of wound healing. Aramwit and Sangcakul52 investigated the application of 8% sericin cream on full-thickness skin wounds on rats. The authors noted complete healing (as confirmed by histology) within 15 days and a significant increase in collagen deposition in comparison with wounds treated with cream.52 Interestingly, wounds treated with cream caused a greater degree of ulceration and infiltration with acute inflammatory exudate 15 days after treatment.52

These results are consistent with in vivo results showing SF biomaterials increase the presence of fibroblasts and subsequent collage deposition in cutaneous rat wounds at days 6 and 12 compared with control and wounds treated with hydrocolloid dressings.52 A supporting study investigating the performance of an SS-based gel formulation further showed decreased inflammatory infiltration and necrosis within dorsal skin flaps in rats compared with the placebo group.53

Surface chemistry also plays a key role in affecting cellular behavior and response.54 Protein adsorption and cell adhesion can be altered by changes in hydrophilicity on SF surfaces.54 The duration of degumming time of SF during scaffold manufacturing is another factor that may have an effect on cell viability.35,47 Tsubouchi et al22 reported a reduction of cell viability following a longer degumming time, potentially attributable to the decomposition of SF following prolonged treatment or the release of small peptide domains.47 Newer studies suggest that cell adhesion and proliferation are affected because prolonged degumming alters the surface chemistry and causes SF protein degradation.35

Within the physiologic process of normal wound healing, it is important to achieve a balance between oxidative stress and antioxidants. Silk sericin is known for its strong antioxidant activity secondary to its amino acid sequence,23 allowing SS to scavenge on free radicals and ROS while increasing the activity of antioxidant enzymes. The hydroxyl side groups of SS also allow the chelating of trace elements.55,56 Further, it has been suggested that SS suppresses epidermal oxidative stress by impeding the production of free radicals secondary to UV irradiation, protecting skin keratinocytes from UV B–induced apoptosis and tumorigenesis.57

Other studies have shown that SS has a mitogenic effect on multiple mammalian cells, including human epithelial cells.21,58 The presence of soluble sericin shortens the lag phase of cell cycle, promoting growth significantly. However, this response is dose dependent; concentrations greater than 1% are harmful to cells.58 Lower-molecular-weight SS peptides (5–100 kDa) also showed a greater ability to promote cell growth than those with higher molecular weight (50–200 kDa).58,59

Collectively, these studies support both SF- and SS-based dressings, both of which can mediate controlled cell infiltration, fibroblast proliferation, collagen deposition, and ROS scavenging. It is worth noting that the method of production of such dressings can also have an overarching role in wound healing.

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Degree of Neovascularization

Neovascularization in skin tissue engineering is a dynamic process involving the distribution of microvessels and the growth of capillaries.60 Neovascularization is essential for adequate wound healing. Patients with comorbidities including diabetes, peripheral vascular disease, and renal insufficiency usually have poor wound healing secondary to impaired neovascularization. In the context of wound healing, neovascularization usually occurs via angiogenesis. The degree of angiogenesis has been investigated in a number of in vivo studies using various SF biomaterials in wound healing.

The prerequisite for angiogenesis is the secretion of vascular endothelial-derived growth factor and fibroblast growth factor (FGF), which promote endothelial budding and vessel formation.60 A study conducted by Baoyong et al43 showed positive expression of FGF cells on the seventh day after operation in a spider silk group compared with no expression of FGF in a negative control group. The expression was mostly found in the epidermis and subcutaneous tissue. This increased secretion from SF is hypothesized to promote vascular proliferation and improve wound healing.

Chouhan et al50 showed that wounds treated with poly (vinyl alcohol) + Antheraea assama SF had very thick granulation tissue and high number of blood vessels on day 7 (Figure 3). This suggested SF materials induced angiogenesis and facilitated the proliferation stage of wound healing earlier than in untreated wounds. Supporting in vitro studies showed the initial formation of small capillary-like structures as early as 7 days.34,40 Functional blood vessels were later observed spreading out proportionally throughout the SF scaffold after 18 days.61

Figure 3

Figure 3

Other studies have specifically investigated the degree of neovascularization from the use of SF films. Zhang et al62 optimized SF films into dermal substitutes then implanted in the back of rats for recovery of dermal tissue loss. Abundant microvessels could be seen as early as 24 hours after implantation. By day 23, the extent of microvasculature and percentage of capillary ingrowth were almost equal to those of normal tissue.62 These findings are supported by recent findings by Wu et al56 with α-smooth muscle actin staining showing an increased number of vessels in the SF film group on day 7 postimplantation in mice; this could promote subsequent skin regeneration.

The number of studies that look at the degree of vascularization after use of SS-based dressings is very limited. Ersel et al53 showed a significant increase in the number of intact vessels within an SS-treated group (applied to a cutaneous wound in an incision skin model in rats) compared with a placebo group. This difference was further associated with an increase in the level of induced nitric oxide synthase staining within the sericin-treated wounds. Interestingly, induced nitric oxide synthase is important in endothelial proliferation and neoangiogenesis.63

Although the above studies show silk materials improve the degree of vascularization in comparison with untreated wounds, the vascularizing abilities of different types of silk biomaterials are heterogeneous. This seems to be dependent on variables including the method of processing and intrinsic biomaterial features such as pore size and interconnectivity.64,65 Numerous studies have been conducted on the use of preseeded silk biomaterials to induce the angiogenic pathway.66 Preseeding scaffolds with endothelial cells or osteogenic cells67 have induced angiogenic factors and endothelial cell organization within in vivo models.

Despite this convincing evidence, no study has investigated the use of preseeded silk biomaterial in the context of wound healing. This may be an exciting future avenue for wound healing studies. In addition, there have been very few human studies conducted to further support the body of evidence of SF film as an accelerator of neovascularization. However, human studies would be challenging because they would require biopsy over a healing or healed wound, which could potentially add to patient-related complications.

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Cytotoxicity/Sensitivity

The ideal wound dressing should be nontoxic and nonimmunogenic and provide a moist healing environment. Various histology and immunohistochemistry studies have shown that SF is biocompatible and has a low immunogenicity.

Multiple in vitro studies have also detected low levels of inflammatory markers produced, mainly tumor necrosis factor α (TNF-α), interleukin 1β, and cyclooxygenase 2 gene.27,36,37,40 The cyclooxygenase 2 gene was observed to be back to baseline levels after 30 hours when macrophages’ response to SF was compared with collagen and tissue culture polystyrene.37 Studies showed that silk-based dressings induce less inflammation when compared with collagen-based scaffolds36,37 or other available dressings.

In vivo models have generally shown SF to be biocompatible. An acute dermal toxicity study conducted in rats68 showed no discernible changes in skin, eyes, mucous membranes, or behavioral pattern throughout the 14-day period of observation after silk protein patch removal. In addition, none of the biochemical parameters measured (aspartate transaminase, alanine transaminase, creatinine, and blood urea nitrogen) differed significantly in the silk biomaterial–treated group from the control group.68 Further skin sensitization studies showed no local signs of erythema, eschar, or edema in the silk-treated group. These findings are consistent with a study conducted by Pavankumar.67 Skin sensitization score was zero (on the Draize scoring system) at all observation time points after application of silk biomaterial on cutaneous wounds in white rabbits.67

The mechanism by which SF may be immunoinhibitory was investigated by Panilaitis et al.37 This study suggested that silk fibers may have an inhibitory effect on the inflammatory response. Lipopolysaccharide-stimulated macrophages produced only up to 10% of TNF-α when cultured in an SF scaffold (Figure 4).37 Antagonism of TNF-α has been shown to increase matrix synthesis, associated with inflammatory suppression through inhibition of downstream nuclear factor κβ binding.69 However, low immunogenicity has only been shown in macroscopic silk fibers based on TNF and ribonuclease protection assay analysis.37 High levels of TNF expression were seen in microscopic, irregularly shaped crystalline fibroin fraction particles approximately 10 to 200 μm.37 This is supported by other studies on polyethylene,55 polymethylmethacrylate,70 and chitin,3 suggesting that particulates in this size range induce macrophage activation.

Figure 4

Figure 4

Table 1

Table 1

Table 2

Table 2

An inflammatory response is seen only when SS is physically associated with the core fibroin fibers.37 Soluble sericin on its own failed to induce the expression and release of TNF-α from macrophages in short- or long-term in vitro studies, suggesting that there may be a synergistic effect with lipopolysaccharides triggering the release of TNF-α when sericin is associated with crystalline fibroin fibers.37

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Human Studies

Despite the positive results of in vivo and in vitro studies, there is still a lack of translation of silk-based polymers into clinical use. The only silk-based product approved by the Chinese Food and Drug Administration is an SF/silicone-based two-layered spongy dressing, licensed for the management of partial- and full-thickness wounds, donor site wounds, and burns. Only one blinded randomized controlled trial has been conducted,63 comparing a pure SF film with this product. Interestingly, results showed that the SF film had better fluid-handling capacity and gas permeability with fewer adverse outcomes. The SF film was also reported to have high transmittance, which is helpful because the skin healing progress could be directly observed and assessed without the need to remove the dressing.

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Limitations

Only 1 randomized controlled trial involving human subjects was identified. The other studies identified in this review were either in vitro or in vivo animal models of wound healing. Although these models have helped to test the application efficacy of SF and SS on certain cell types including fibroblasts and keratinocytes, it is difficult to extrapolate these findings into clinical practice in humans because of anatomical and physiological variances. To do so, definitive studies must be conducted on human subjects, although this requires consideration of practical, ethical, and moral barriers.

A further limitation to the studies enlisted in this review is outcome reporting bias. Without temporal biopsies taken throughout the wound healing process, it is difficult to accurately define the wound by histology. The majority of the studies included in this review do not use this analytical technique to define the healing wound. Although this method poses obvious practical issues, it may be necessary as a further stepping stone to human studies.

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CONCLUSIONS

Silk-based dressings have been studied extensively in the context of wound healing using in vitro and in vivo models. The literature suggests they show good biocompatibility and wound healing properties compared with commonly used dressings and possess a low toxicity profile. Silk fibroin promotes excellent collagen deposition within in vivo animal models of full-thickness wounds. Likewise, SS provides good structural support for cell attachment and migration, accelerating wound healing. However, in comparison to SF, SS has less antimicrobial properties. Despite the promise of SS and SF as wound healing dressings, only a single randomized controlled trial has been conducted in patients. These authors encourage further study to investigate the wound healing properties of SF and SS within humans.

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Keywords:

cutaneous injury; silk fibroin; silk sericin; tissue regeneration; wound dressing; wound healing

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