Microstructure of the Silk Fibroin-based Hydrogel Scaffolds Derived from the Orb-web Spider Trichonephila clavata

doi:10.21203/rs.3.rs-3620157/v1

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Microstructure of the Silk Fibroin-based Hydrogel Scaffolds Derived from the Orb-web Spider Trichonephila clavata

https://doi.org/10.21203/rs.3.rs-3620157/v1

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published 10 Feb, 2024

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Due to the unique properties of the silk fibroin (SF) from silkworm the SF-based hydrogels recently received significant attention for various biomedical applications. However, researches regarding the SF-based hydrogels isolated from spider silks have been comparatively restricted due to shortage of collection and preparation of naïve silk materials. Therefore, this study focused on the microstructural characteristics of hydrogel scaffolds derived from two types of woven silk glands – major ampullate gland (MAG) and tubuliform gland (TG) - in the orb-web spider Trichonephila clavata and compared them with those of silk fibroin (SF) hydrogel scaffold extracted from the cocoon of the insect silkworm Bombyx mori. Our FESEM analysis revealed that the SF hydrogel has high porosity, translucency, and a loose upper structure, with attached SF fibers providing stability. The MAG hydrogel displayed even higher porosity, elongated fibrous structures, and improved mechanical properties, while TG hydrogel showed increased porosity, ridge-like or wall-like structures, and stable biocapacity is formed by physical cross-linking. The distinct microstructural characteristics of MAG and TG hydrogels are expected to provide advantages in the selection of tailored substrates to support specific cell types for tissue engineering and regenerative medicine applications.

microstructure

hydrogel

scaffold

silk

spider

Hydrogel is an example of an elastic gel, formed from a polymer compound solution (Khademhosseini & Demirci, 2016). It uses water as a dispersion medium, has the ability to absorb and retain a large amount of water, and possesses a cross-linked network structure (Tamura et al., 2000). This cross-linking occurs through valence bonds, hydrogen bonds, and other mechanisms, resulting in a polymer electrolyte with a three-dimensional structure (Xu et al., 2007). The abundance of water in the hydrogel allows substances dissolved within it and substances with relative molecular mass to permeate and diffuse ((Liu & Fan, 2005; Gulrez et al., 2011; Ahmed, 2015).

The unique structure of hydrogels contributes to their excellent properties, making them an ideal scaffold material in tissue engineering repair (Prince et al., 1995; Teulé et al., 2012). The aqueous surroundings of hydrogels offer protective advantages for both cells and small molecule drugs (Yu et al., 2016). Additionally, these hydrogels promote cell adhesion and proliferation, fostering biological processes (Baroli, 2007). Certain hydrogel types even allow for in situ injection, enhancing their versatility (Shin et al., 2019). The inherent stability of hydrogel shapes, coupled with their uncomplicated fabrication process, ensures ease of handling (Chai et al., 2017).

Over the course of more than 5,000 years, silk has been extensively studied and utilized, while spider silk has become a hot topic in material science in the past decade. Silk protein is a natural protein polymer secreted by arthropods such as insects and spiders (Belbéoch et al., 2021). Both types of natural silks exhibit high mechanical properties, including elasticity and tensile strength, as well as exceptional biological adaptability. Spider silk, in particular, surpasses chemical fibers like aramid and nylon known for their strength (Hardy & Scheibel, 2010).

These proteins are synthesized in epithelial cells and stored in the glands until they are spun into fibers through spinning tubes or other ducts, resulting in natural materials with unique functions (Moon, 2018; Altman et al., 2003). Spider silk fibers consist of multiple spider fibrils formed by β-sheets combined with amorphous regions. The primary amino acids present in spider silk are alanine, glycine, and serine, with alanine providing strength and glycine imparting elasticity to spider silk (Römer & Scheibel, 2008; Kiseleva et al., 2020).

Spider silk stands out as a remarkable biopolymer with exceptional chemical, physical, and biological properties (Blamires et al., 2017). Compared to other biopolymers such as collagen, polylactic acid, fibrin, alginate, and gelatin, spider silk possesses unique biomolecular properties (Withanage et al., 2021). It significantly reduces inflammatory responses, acts as a scaffold for various cell types' growth, and promotes cell adhesion, making it superior to other biopolymers (Li et al., 2015; Lee et al., 2016; Salehi et al., 2020). In recent times, spider silk has found extensive use in biomaterials such as films, matrices, and hydrogels, leading to numerous applications in biomedicine (Johansson et al., 2015; Stoppel et al., 2017; Borkner et al., 2019).

The golden orb web spider, Trichonephila clavate, a member of the Trichonephila genus, belongs to the Araneidae family and serves as a subgenus of Nephila (Kuntner et al., 2019). These spiders have seven distinct types of silk-producing glands (Park & Moon, 2014), each responsible for producing silk with specific compositions and mechanical properties to fulfill biological functions (Eisoldt et al., 2011; Sun et al., 2023).

Despite the increasing use of spider silk as a biomaterial, obtaining large quantities of natural spider silk remains challenging, and the production of artificial spider silk poses several difficulties (Bakhshandeh et al., 2021). In this study, we utilized field emission scanning electron microscopy (FESEM) to examine dragline and cocoon silk-derived hydrogel scaffolds produced from the MAG and TG of T. clavate spiders. By comparing the microstructure of these scaffolds with silk-based ones, we hope to gain valuable insights into the future applications of natural spider silk in biomaterials.

The experimental orb-web spider Trichonephila clavata, belonging to the family Aranidae: Nephilidae, was used in this study. The spiders were collected near Dankook University, Cheonan, Chungnam, South Korea. The genus Trichonephila, previously considered a subgenus of Nephila, was elevated to the genus level (Kuntner et al., 2019). Captured spiders were kept in wooden frames (40 × 40 × 10 cm) with acrylic panels on the front and rear sides, providing natural lighting. The spiders were fed insects and water daily.

To obtain two spider silk gland samples, carbon dioxide was injected into a sealed vial containing an adult female spider. After 30 seconds, the spider was anesthetized and dissected under a light microscope. The spiders were then placed in a spider’s Ringer solution which consisted of 160 mM NaCl, 7.5 mM KCl, 4 mM CaCl₂, 1 mM MgCl₂, 4 mM NaHCO₃, 20 mM glucose, and had a pH of 7.4 (Moon, 2018; Moon and Tillinghast, 2020), and the major ampullate gland (MAG) and tubular gland (TG) were carefully removed. The glands were ground with dissecting forceps to fully collect the spider silk protein droplets. The collected liquid was transferred to a 2 mL centrifugal tube, centrifuged at 3000 rpm for 30 seconds, and the supernatant was stored frozen at -20°C.

To prepare the silk fibroin (SF) solution for hydrogel production, cocoons weighing 150 g were cut into small pieces and subjected to degumming three times in 0.05% (w/w) Na₂CO₃ solution at 100°C for 30 minutes. The fibers were thoroughly rinsed and dried in an oven. The extracted silk fibroin (SF) fibers were then dissolved in a ternary solvent of CaCl2/CH3CH2OH/H2O (molar ratio 1:2:8) at 70 ± 2°C for 4 hours (Ajisawa, 1998). The SF solution was obtained after dialysis and filtration.

The SF hydrogel was formed by pouring the SF solution into a mold, vortexing at 9000 rpm for 7 minutes, and then incubating the mold at 37°C for 1–2 hours. The MAG hydrogel scaffold was created by mixing the spider MAG silk solution with the same silkworm SF solution at a rating of 1: 3, followed by the MAG hydrogel formation process as the SF hydrogel. TG hydrogels Scaffold using the spider TG silk solution, mixed with the silkworm of solution at a ratio of 1:3. The mixed solution was vortexed at 9000 rpm for 7 minutes and then incubated in a 37°C incubator for 1–2 hours.

After preparation, the three hydrogels were immersed in a freeze-drying device set at -80°C and a pressure of 80 mmHg for 48 hours to prepare observation samples (FDCF-12012, Oufeilong Company, Shanghai, China). The microstructural analysis of the hydrogels was performed using field emission scanning electron microscopy (FESEM). The samples were coated with a 20 nm layer of platinum-palladium using a Hitachi E-1030 ion sputter coater, and FESEM (S-4300, Hitachi Co., Tokyo, Japan) was used to observe the samples at an accelerating voltage of 5–20 kV (Sun et al. 2020, 2023)

Through visual observation, it is evident that the SF hydrogel produced in this experimental endeavor possesses a distinctly translucent quality. Notably, the uppermost layer of the hydrogel exhibits a conspicuously more relaxed structure. This augmented looseness in the structure can be reasonably ascribed to the vigorous shaking process employed during the initial stages of forming the aqueous SF solution (Fig. 1A). MAG hydrogels display a distinct yellowish translucence attributed to the presence of yellow protein droplets generated by the MAG of T. clavata.

Additionally, the formation process, characterized by a vortex within the protein solution, leads to a visibly less compact structure in the upper layer of the MAG hydrogels (Fig. 1B). Given that the TG of T. clavata generates protein droplets with a white hue, the resulting TG hydrogel presents a milky white and translucent appearance to the unaided eye. Furthermore, upon creating the hydrogel through vortexing the protein solution, a discernible distinction can be made with the naked eye: the upper layer of the TG hydrogel exhibits a visibly looser structure compared to the lower layer (Fig. 1C).

Following the freeze-drying of the SF solution at -80°C, we conducted a detailed examination of the resulting hydrogels using Field Emission Scanning Electron Microscopy (FESEM). The hydrogel unveiled a striking high-porosity network structure characterized by ridge-like or wall-like formations. The fracture after drying of the hydrogel was sharp and the cross-section was uneven (Fig. 2A). Notably, the average porosity of the silk scaffolds registered at an impressive 52.1%, underscoring their remarkably porous nature. The hydrogel scaffold exhibited a pore-like architecture with an approximate diameter of 59 µm and pore wall thickness spanning from 4.5 to 6.5 µm (Fig. 2B).

This intricate porous structure facilitates efficient material exchange and serves as an optimal conduit for nutrient transport in the context of in vitro cell culture. Upon closer examination at higher magnification, we discerned block-like structures adhering to the hydrogel wall, likely remnants of small hydrogel fragments that detached during the drying process, measuring approximately 10.0 ~ 13.5 µm in length (Fig. 2C). Additionally, when observed at a higher magnification level, the surface of the hydrogel wall exhibited a relatively rough texture, marked by clusters of SF fibers aggregated together. This particular structural attribute augments surface friction and promotes cell adhesion during cell culture (Fig. 2D).

Similarly, following the freeze-drying process at -80°C, we employed FESEM to examine the morphological features of MAG hydrogels, created by combining spider MAG protein solution and SF at a volume ratio of 1:3. The microstructure of these hydrogels also reveals a network configuration characterized by heightened porosity and the presence of ridge or wall-like structures (Fig. 3A).

Furthermore, it is worth noting that the fracture appearance of the hydrogel after drying is sharper, while the cross-section appears flatter in comparison to that of the SF hydrogel. This architectural design promotes efficient material exchange and functions as an excellent conduit for nutrient transport in the context of in vitro cell culture, providing cells with a medium characterized by increased porosity. In comparison to SF gels, the pore structure of MAG hydrogels exhibits greater uniformity in size, with pores distributed more evenly across the pore wall structure (Fig. 3B).

Notably, the average porosity of the MAG hydrogel scaffold stands at 62.6%, which is 1.2 times that of the SF hydrogel scaffold. This elevated porosity enhances nutrient transport and facilitates intercellular nutrient exchange. The diameter of the pore-like structures on the hydrogel scaffold measures approximately 48 µm. However, MAG hydrogels feature thicker pore walls, ranging from 3.5 to 5.0 µm, resulting in enhanced mechanical properties relative to SF hydrogels (Fig. 3C).

Upon closer examination under high magnification, the wall surface of the hydrogel exhibits a relatively rough texture. When scrutinizing the hydrogel wall through high magnification FESEM, slender fiber structures adhering to the hydrogel wall become apparent, with a fiber length measuring approximately 10 to 30 µm. This structural feature increases surface friction and facilitates cell adhesion during cell culture (Fig. 3D).

We also conducted FESEM analysis to examine the morphological characteristics of freeze-dried TG hydrogels, which were created by blending spider TG protein solution and SF at a volume ratio of 1:3. In comparison to SF, the TG hydrogel manifests a network structure with elevated porosity, featuring ridge or wall-like formations (Fig. 4A). It is noteworthy that the fracture appearance of the dried hydrogel wall appears less sharp than that of the other two hydrogels, and the cross-section exhibits a relatively smooth profile. Moreover, in contrast to the MA hydrogel, the pores in the TG hydrogel appear more dispersed, lacking the regular shape seen in MA hydrogels and displaying non-uniform sizes (Fig. 4B).

The average porosity of the TG hydrogel scaffold measures at 52.70%, positioning it between the porosity values of SF hydrogel and MAG hydrogel. This observation suggests potential future applications based on the distinct porosity levels among these three hydrogels. The diameter of the pore-like structures on the hydrogel scaffold is approximately 53 µm. The thickness of the pore walls ranges from 1.5 to 3.5 µm (Fig. 4C), making it the thinnest among the three hydrogels and reflecting the weakest relative mechanical properties. Upon closer examination using high-magnification FESEM, we observed that the hydrogel wall appeared smooth, devoid of any attached fibrous structures (Fig. 4D).

Through examination of the three hydrogels using Field Emission Scanning Electron Microscopy (FESEM), it becomes possible to formulate a simplified model diagram based on their respective findings. This diagram serves to enhance comprehension of the pore characteristics inherent in these hydrogels refer to Fig. 5. The SF hydrogel scaffold displays a porous structure with pore diameters approximately measuring 59 µm and pore wall thicknesses spanning from approximately 4.5 to 6.5 µm (Fig. 5A).

Conversely, the MAG hydrogel scaffold features pore-like structures with a diameter of about 48 µm, slightly smaller than that of the SF hydrogel, constituting roughly 80% of its pore diameter. Notably, the MAG hydrogels exhibit thicker pore walls within the range of 3.5 to 5.0 µm, resulting in heightened mechanical properties on a relative scale (Fig. 5B).

As for the TG hydrogel scaffold, the diameter of its pore-like structures measures around 53 µm, again slightly less than that of the SF hydrogel yet approximately 1.1 times greater than that of the MAG hydrogel. Interestingly, the pore walls of the TG hydrogel span a thickness range of 1.5 to 3.5 µm, rendering it the thinnest among the three hydrogels and consequently displaying the least robust relative mechanical properties (Fig. 5C).

FESEM imaging of the lyophilized hydrogel revealed the fibril structure, and the crosslinked fibril network confirmed the successful chemical crosslinking between silk proteins and spider silk proteins, resulting in the formation of the hydrogel network. Among the three hydrogels, the MAG hydrogel had the smallest pore size, while the SF hydrogel had the largest average pore size (Table 1).

Table 1

Structural characteristics of three types of the hydrogel scaffolds
Hydrogel	Porosity (%)	Diameter (µm)	Thickness (µm)
SF MAG TG	52.1 62.6 52.7	59 48 53	4.5–6.5 3.5–5.0 1.5–3.5

* MAG: major ampullate gland, SF: silkworm fibroin, TG: tubuliform gland

Hydrogels are three-dimensional network polymers with high water content (more than 95% w/w) and high swelling rates (Rammensee et al., 2006; Yan et al., 2006). They utilize water as the dispersion medium, can absorb and retain a large amount of water, and possess a cross-linked network structure. The cross-linking is achieved through valence bonds, hydrogen bonds, and other interactions, resulting in polymer electrolyte characteristics and a three-dimensional structure (Kopeček, 2007; Kabiri et al., 2011).

Fibroin hydrogels, in particular, are formed by combining silk fibroin (SF) molecules through non-covalent bonds, such as hydrogen bonds, hydrophobic group interactions, electrostatic interactions, ionic interactions, and chain entanglements. These physically cross-linked formulations make SF hydrogels highly biocompatible and biodegradable, making them promising candidates for various applications, especially in the biomedical field (Zheng & Zuo, 2021).

Pore size and porosity stand as fundamental parameters when assessing the structural characteristics of hydrogels. Porosity, which refers to the proportion of empty space within a solid material, constitutes a material-independent morphological property that bears significant importance in the realm of bone tissue formation (Kuboki et al., 1998). A heightened level of porosity fosters cellular proliferation by easing the passage of oxygen and nutrients through larger pore spaces, conversely, scaffolds featuring lower porosity can enhance specific cellular functions, such as osteogenic cultures, while retarding cell proliferation and aggregation (Takahashi & Tabata, 2004).

Within the scope of this study, the analysis of SF hydrogels unveiled a network structure characterized by robust porosity and a ridge- or wall-like formation that facilitates efficient material exchange and nutrient transport. Notably, SF fibers were observed to adhere to the hydrogel's walls, thus bolstering its structural stability. The average porosity of SF hydrogel measures at 52.1%, rendering it translucent with a relatively loose upper structure. Similarly, MAG hydrogels exhibit a network structure with even higher porosity and a ridge- or wall-like configuration, actively promoting efficient material exchange and nutrient transport during in vitro cell culture (Karageorgiou & Kaplan, 2005).

Additionally, elongated fibrous structures were identified adhering to the hydrogel walls, further fortifying its structural integrity. MAG hydrogel assumes a yellow translucent appearance and possesses a loose upper structure, with an average porosity 1.2 times higher than that of SF hydrogels, indicative of enhanced nutrient transport (Takahashi & Tabata, 2004).

Distinctively, TG hydrogels feature elevated porosity levels and showcase unique ridge- or wall-like structures. Unlike SF and MAG hydrogels, TG hydrogels do not host attached fibrous structures on their walls. They present as milky and translucent, boasting unique pore diameters and relatively thin pore walls, thereby yielding distinctive mechanical properties. These findings align with those reported by Liu et al. in 2019, reaffirming the successful chemical cross-linking between silk and spider silk proteins, which in turn validates the formation of a robust hydrogel network.

Our recent FESEM analysis has proven instrumental in elucidating the morphological traits of SF, MAG, and TG hydrogels. SF hydrogels, characterized by attached SF fibers, exhibit noteworthy porosity, while MAG hydrogels manifest even higher porosity levels, elongated fiber structures, and superior mechanical attributes. TG hydrogels, on the other hand, exhibit augmented porosity coupled with a distinctive ridge- or wall-like structure, signifying successful chemical cross-linking. In the context of cell culture, a study by Yuan et al. in 1999 suggested that the roughness and high porosity of MAG hydrogels render them more conducive to cellular proliferation.

Nevertheless, the selection of a hydrogel should be undertaken with due consideration for the specific requisites of distinct cellular tissues in both in vitro and in vivo settings (Zou et al., 2022). Our recent results of the morphological properties of these hydrogels holds the potential to illuminate their applications in tissue engineering, drug delivery, and regenerative medicine. It is expected that other subsequent research work can be based on the results obtained in this study to further optimize its performance and expand its practical applications.

When it comes to classifying SF hydrogels based on porosity, they fall into two primary categories: macropores and nanopores (Isobe et al., 2018). Macroporous SF hydrogels feature larger pore sizes that are discernible to the naked eye or under an optical microscope, typically ranging from tens to hundreds of microns. Their porosity values typically span from 50–90%. In contrast, nanoporous SF hydrogels possess much smaller pore sizes, usually less than 100 nm (Ak et al., 2013; Isobe et al., 2018).

The specific pore characteristics of SF hydrogels assume a pivotal role in determining their suitability for various applications. Consequently, in line with the findings of Karageorgiou & Kaplan (2005), the hydrogel acquired in this study displayed macroporous attributes, facilitating the unhindered flow of nutrients, oxygen, and cells within the hydrogel matrix. It is worth noting that the smaller pore size of MAG hydrogels may constrain cellular penetration and migration within the hydrogels. However, it is plausible to anticipate that by adjusting the MAG protein-to-SF ratio, hydrogels with varying pore sizes can be tailored to meet specific requirements.

Moreover, the thicker pore walls observed in MAG hydrogels contribute to enhancing their mechanical properties, rendering them suitable for applications where mechanical strength is a critical consideration. Conversely, the augmented porosity in TG hydrogels, as predicted based on the research results of Isobe et al. (2018), promises to facilitate efficient material exchange and nutrient transport.

Large-pore silk fibroin hydrogels derived from spider silk proteins exhibit distinctive pore characteristics, wall thickness, and porosity, making them well-suited for loading and controlling the release of therapeutic agents such as growth factors, proteins, and drugs, (Schacht et al., 2015). These hydrogels hold substantial potential as delivery systems for achieving localized and sustained drug release in applications within the domains of tissue engineering and regenerative medicine.

Physical crosslinking involves the transformation of SF molecules from a random coil structure to a β-sheet conformation by manipulating specific physical factors, ultimately leading to further aggregation and the formation of a hydrogel with a three-dimensional network structure (Koh et al., 2015; Aigner et al., 2018). The β-sheet content within the SF solution significantly impacts pore size, distribution, and mechanical properties (Kim et al., 2004; Vepari & Kaplan, 2007; Slotta et al., 2008).

The relatively looser structure observed in the upper layer of SF hydrogel may affect its mechanical properties and limit its applicability in scenarios demanding a more uniform structure (Widhe et al., 2016). In contrast, the thicker pore walls of MAG hydrogels contribute to their enhanced mechanical properties, rendering them suitable for applications where mechanical strength is paramount. Moreover, the ridge-like or wall-like structure of MAG hydrogels provides efficient nutrient transport channels, which prove advantageous for in vitro cell culture. However, compared to SF and MAG hydrogels, TG hydrogels exhibit relatively thin pore walls, resulting in weaker mechanical properties and limiting their use in applications requiring higher mechanical strength.

The β-sheet content in the SF solution influences pore size, distribution, and mechanical characteristics (Koh et al., 2015). On the other hand, spider silk proteins self-assemble into β-sheet-rich nanofibers through the nucleation-aggregation mechanism to achieve gelation, with concentration playing a pivotal role in this process (Römer & Scheibel, 2008; Kiseleva et al., 2020).

The distinct morphological characteristics of SF, MAG, and TG hydrogels each offer advantages and disadvantages for various applications. SF hydrogels boast high porosity and stability, albeit with a potentially looser structure. MAG hydrogels exhibit higher porosity, improved mechanical properties, and efficient nutrient transport, though they have smaller pore sizes and a yellow appearance. TG hydrogels feature increased porosity, successful chemical crosslinking, and unique morphological attributes, but their mechanical properties are comparatively weaker, and they have a milky white appearance.

The silkworm cocoon, spider egg sac, and spider dragline silk can be effectively employed for chondrocyte culture in vitro (Gellynck et al., 2008). Consequently, it is reasonable to infer that the hydrogel scaffolds generated in this study hold great potential for future biomedical applications. These scaffolds can be adapted to diverse culture settings, offering tailored substrates with varying porosity and other distinguishing characteristics to best support specific cell types. A comprehensive understanding of these properties is essential when selecting the most suitable hydrogel for specific applications in fields such as tissue engineering, regenerative medicine, and drug delivery.

Researches on SF-based hydrogels isolated from spider silk has been relatively restricted due to the shortage of collection and preparation of naive silk materials. Therefore, we focused on the microstructural characteristics of hydrogel scaffolds derived from the major ampullate gland (MAG) and tubuliform gland (TG) of the spider T. clavata. Additionally, it was compared with a silk fibroin (SF) hydrogel scaffold extracted from cocoon of the silkworm (B. mori) cocoon. FESEM analysis showed that hydrogels derived from spider silk glands exhibited relatively higher porosity, elongated fibrous structure, and improved mechanical properties than SF hydrogels from silkmoth alone. The microstructural characteristics of MAG and TG hydrogels are expected to provide advantages in substrate selection for tissue engineering and regenerative medicine applications.

FESEM: field emission scanning electron microscopy
MAG: major ampullate gland
SF: silk fibroin
TG: tubuliform gland

Availability of data and materials

Materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes

Competing interests

Not applicable

Funding

This research was supported by the National Research Foundation (NRF) of Korea funded by the Korea government (MSIT) (NRF-2019R1I1A3A01062105)

Authors' contributions

Myung-Jin MOON (Corresponding Author): contributions to the conception, design of the work as well as contribution to the acquisition, analysis, and interpretation of data. Yan SUN (First Author): contribution to analysis, and interpretation of data., Bon-Jin KU (Second Author): contribution to preparing manuscript and preparing poster presentation.

Acknowledgements

This research was supported by the National Research Foundation (NRF) of Korea funded by the Korea government (MSIT) (NRF-2019R1I1A3A01062105)

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Microstructure of the Silk Fibroin-based Hydrogel Scaffolds Derived from the Orb-web Spider Trichonephila clavata

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