De Novo Long-Read Genome Assembly and Annotation of the Luna Moth (Actias luna) Fully Resolves Repeat-Rich Silk Genes.

We present the first long-read de novo assembly and annotation of the luna moth (Actias luna) and provide the full characterization of heavy chain fibroin (h-fibroin), a long and highly repetitive gene (>20 kb) essential in silk fiber production. There are >160,000 described species of moths and butterflies (Lepidoptera), but only within the last 5 years have we begun to recover high-quality annotated whole genomes across the order that capture h-fibroin. Using PacBio HiFi reads, we produce the first high-quality long-read reference genome for this species. The assembled genome has a length of 532 Mb, a contig N50 of 16.8 Mb, an L50 of 14 contigs, and 99.4% completeness (BUSCO). Our annotation using Bombyx mori protein and A. luna RNAseq evidence captured a total of 20,866 genes at 98.9% completeness with 10,267 functionally annotated proteins and a full-length h-fibroin annotation of 2,679 amino acid residues.

Silk Fibroin Microneedles Loaded with Lipopolysaccharide-Pretreated Bone Marrow Mesenchymal Stem Cell-Derived Exosomes for Oral Ulcer Treatment.

Oral ulcers, superficial lesions on the surface of the oral mucosa, have a high incidence rate, and their main symptoms include local pain and erosion. Lipopolysaccharide (LPS)-preconditioned bone marrow mesenchymal stem cells and their secreted exosomes (LPS-pre-Exos) have been shown to promote recovery in various inflammatory conditions and wounds. However, studies documenting LPS-pre-Exos as a therapeutic intervention for oral mucosal-like diseases are lacking. In this study, we prepared a silk fibroin microneedle (MN) patch consisting of LPS-pre-Exos and zeolitic imidazolate framework-8 (ZIF-8) that localized at the tip and base, respectively, and used this MN patch for oral ulcer treatment. Upon insertion into the oral mucosa, continuous LPS-pre-Exos release was observed, which promoted macrophage polarization and tissue healing. Additionally, the ZIF-8 framework in the MN patch facilitated the controlled release of Zn2+, which demonstrated potent antimicrobial properties via synergistic effects. The in vitro experimental results showed that the silk fibroin MN patch can continuously release LPS-pre-Exos and Zn2+ for more than 7 days. Thus, the LPS-pre-Exos and ZIF-8-loaded silk fibroin MN patch exhibited good anti-inflammatory and antibacterial properties, promoting oral ulcer healing, and showed good histocompatibility. Hence, it may represent a potentially valuable strategy for facilitating oral ulcer healing.

Injectable vancomycin-loaded silk fibroin/methylcellulose containing calcium phosphate-based in situ thermosensitive hydrogel for local treatment of osteomyelitis: Fabrication, characterization, and in vitro performance evaluation.

The conventional treatment of osteomyelitis with antibiotic-loaded nondegradable polymethylmethacrylate (ATB-PMMA) beads has certain limitations, including impeded bone reconstruction and the need for secondary surgery. To overcome this challenge, this study aimed to develop and characterize an injectable vancomycin-loaded silk fibroin/methylcellulose containing calcium phosphate-based in situ thermosensitive hydrogel (VC-SF/MC-CAPs). The VC-SF/MC-CAPs solution can be easily administered at room temperature with a low injectability force of ≤30 N and a high vancomycin (VC) content of ~96%. Additionally, at physiological temperature (37 °C), the solution could transform into a rigid hydrogel within 7 minutes. In vitro drug release performed under both physiological (pH 7.4) and infection conditions (pH 4.5) revealed a prolonged release pattern of VC-SF/MC-CAPs following the Peppas-Sahlin kinetic model. In addition, the released VC from VC-SF/MC-CAPs hydrogels exhibited antibacterial activity against Staphylococcus aureus for a period exceeding 35 days, as characterized by the disk diffusion assay. Furthermore, at pH 7.4, the VC-SF/MC-CAPs demonstrated >60% degradation within 35 days. Importantly, when exposed to physiological pH conditions, CAPs are transformed into bioactive hydroxyapatite, which benefits bone formation. Therefore, VC-SF/MC-CAPs showed significant potential as a local drug delivery system for treating osteomyelitis.

Overview and Evolution of Insect Fibroin Heavy Chain (FibH).

The FibH gene, crucial for silk spinning in insects, encodes a protein that significantly influences silk fiber mechanics. Due to its large size and repetitive sequences, limited known sequences of insect FibH impede comprehensive understanding. Here, we analyzed 114 complete FibH gene sequences from Lepidoptera (71 moths, 24 butterflies) and 13 Trichoptera, revealing single-copy FibH in most species, with 2-3 copies in Hesperinae and Heteropterinae (subfamily of skippers). All FibH genes are structured with two exons and one intron (39-45 bp), with the second exon being notably longer. Moths exhibit higher GC content in FibH compared to butterflies and Trichoptera. The FibH composition varies among species, with moths and butterflies favoring Ala, Gly, Ser, Pro, Gln, and Asn, while Trichoptera FibH is enriched in Gly, Ser, and Arg, and has less Ala. Unique to Trichoptera FibH are Tyr, Val, Arg, and Trp, whereas Lepidoptera FibH is marked by polyAla (polyalanine), polySer (polyserine), and the hexapeptide GAGSGA. A phylogenetic analysis suggests that Lepidoptera FibH evolved from Trichoptera, with skipper FibH evolving from Papilionoidea. This study substantially expands the FibH repertoire, providing a foundation for the development of artificial silk.

Studies on the potential risk of amyloidosis from exposure to cultured fibril from silk fibroin.

Amyloid A (AA) amyloidosis is induced by administering amyloid fibrils to animals under inflammatory conditions. Silk fibroin (SF), the main component of silk threads, forms amyloid-like fibrils and has been previously reported to induce AA amyloidosis in mice. In this study, SF was cultured in ethanol solution, and after confirming fibril formation through thioflavin T assay, Congo red assay, and observation under electron microscopy, cultured SF ethanol solutions were administered to mice via various routes to investigate the induction of target organs and amyloidosis. As a result, cultured SF ethanol solutions were confirmed to reach the lungs and spleen, but no amyloid deposition was observed. While SF forms amyloid-like fibril structures through cultivation in ethanol solution, its amyloid-enhancing factor (AEF) activity is considered low in mice.

Enhanced Tissue Regeneration Through Immunomodulatory Extracellular Vesicles Encapsulated in Diversified Silk-Based Injectable Hydrogels.

Mesenchymal stem cells (MSCs) immunologically trained using lipopolysaccharide (LPS) display enhanced immunomodulatory capabilities. Extracellular vesicles (EVs) derived from MSCs are widely used in regenerative medicine owing to their bioactive properties without the drawbacks of cell therapy. However, it remains unclear whether EVs derived from LPS-stimulated (trained) MSCs (L-EVs) inherit the enhanced reparative potential from their parent cells. Thus, this study first aims to explore the effect of immunological training on the bioactivity of L-EVs. LPS-trained bone marrow-derived MSCs (BMSCs) secrete more EVs, and these EVs significantly promote M2 macrophage polarization. Subsequently, hydrogel systems based on thixotropic injectable silk fibroin are prepared for in vivo EV delivery. These hydrogels have controllable gelation time and exhibit outstanding reparative effects on rat skin wounds and alveolar bone defects. Finally, it is revealed that L-EVs promote M2 macrophage polarization by inhibiting the nuclear translocation of PKM2. Overall, this study shows that the immunological training of BMSCs effectively improves the therapeutic effects of their EVs and provides a convenient and diversified EV delivery strategy using an injectable silk fibroin hydrogel. This strategy has broad clinical application prospects for tissue regeneration.

Silk fibroin/vitreous humor hydrogel scaffold modified by a carbodiimide crosslinker for wound healing.

Natural-derived biomaterials can be used as substrates for the growth, proliferation, and differentiation of cells. In this study, bovine vitreous humor as a biological material was cross-linked to silk fibroin with different concentration ratios to design a suitable substrate for corneal tissue regeneration. The cross-linked samples were evaluated with different analyses such as structural, physical (optical, swelling, and degradation), mechanical, and biological (viability, cell adhesion) assays. The results showed that all samples had excellent transparency, especially those with higher silk fibroin content. Increasing the ratio of vitreous humor to silk fibroin decreased mechanical strength and increased swelling and degradation, respectively. There was no significant difference in the toxicity of the samples, and with the increase in vitreous humor ratio, adhesion and cell proliferation increased. Generally, silk fibroin with vitreous humor can provide desirable characteristics as a transparent film for corneal wound healing.

Use of Silk Fibroin Material Composites for Green, Flexible Supercapacitors.

Within the context of seeking eco-friendly and readily available materials for energy storage, there is a pressing demand for energy storage solutions that employ environmentally sustainable, high-performance, and adaptable constituents. Specifically, such materials are essential for use in wearable technology, smart sensors, and implantable medical devices, whereas, more broadly, their use plays a pivotal role in shaping their efficiency and ecological footprint. Here, we demonstrate an entirely biopolymer-based supercapacitor with a remarkable performance, achieving a capacitance greater than 0.2 F cm-2 at a charge-discharge current of 10 mA cm-2 with 94% capacitance retention after 20,000 cycles. The supercapacitor is composed of three distinct silk fibroin (SF) composite materials, namely, photo-cross-linkable SF (Sil-MA) hydrogel, SF-polydopamine (SF-PDA), and SF bioplastic, to create a gel electrolyte, electrode binder, and encapsulation, respectively. Together, these elements form a mechanically and electrochemically robust skeleton for biofriendly energy storage devices. Moreover, these biomaterial-based supercapacitor devices show stretchability, flexibility, and compressibility while maintaining their electrochemical performance. The biomaterials and fabrication techniques presented can serve as a foundation for investigating various aqueous electrochemical energy storage systems, especially for emerging applications in wearable electronics and environmentally friendly material systems.

Magnetic-navigable silk fibroin microneedles for oral drug delivery: Ensuring long-lasting helicobacter pylori eradication and rapid hemostasis in the stomach.

The Helicobacter pylori infection in the stomach is the key reason for gastric mucosal bleeding. Eliminating gastric Helicobacter pylori by oral treatment remains difficult due to the presence of the gastric mucosal layer, which acts as a physical barrier to drugs via oral administration. In this study, a magnetic-navigable microneedle drug delivery platform (MNsD) for oral administration, featuring differential dual-mode drug release rate, was designed to fulfil rapid gastric hemostasis and overcome the gastric barriers for long-lasting Helicobacter pylori inhibition in stomach. MNs-D was created by rationally loading the carrier substrate, which was composed of silk fibroin with variable solubility, with antibiotics and hemostats. In vitro experiments showed MNs-D may sustainably eradicate Helicobacter pylori in stimulated gastric juices with long-lasting drug release (79 % in 24 h) and quickly establish hemostasis with instant drug release (92 % within 60 s). Most importantly, in vivo studies demonstrated MNs-D overcame the unsettling gastric mucosal barrier in traditional therapies of oral administration by insertion into the GML under magnetic navigation, resulting in sustained antibiotic release for long-lasting Helicobacter pylori eradiation (99 %). For differential dual-mode medication release against gastric Helicobacter pylori infections, this study may have firstly examined the effects of magnetic navigated microneedles administered orally.

Silk fibroin wetting stability film induced by polyamide-amine-epichlorohydrin (PAE) for intelligent sensing system.

Protein materials gain new functions and applicability through redesigns in protein structure and engineering confer. However, the application and development of proteins for use in flexible devices that fit in flexible devices that fit the surface of human skin is hindered by their poor wet stability. Here, we described the design of wet-stable materials based on the reconstruction of silk fibroin (SF). The combination of polyamide-amine-epichlorohydrin (PAE) was used as a traction rope to bring SF molecular chains closer to each other, to facilitate the self-assembly of SF through branching and lengthening of molecular chains, and change its crystalline structure. SF/PAE composite films that exhibited huge improvement in ductility and wet stability were combined with flexible SF substrates via patterning and ion sputtering to prepare flexible sensors. In addition, the SF/PAE sensing system equipped with a microprocessor and Bluetooth module enabled the real-time remote acquisition of human health signals such as vocal cords, joints, pulse and meridians. This reconfiguration of the SF structure will advance the systematic exploration of protein structures and the development of protein materials for intelligent device applications.

Silk-derived sericin/fibroin mixture drink fermented with plant-derived Lactococcus lactis BM32-1 improves constipation and related microbiota: a randomized, double-blind, and placebo-controlled clinical trial.

We previously showed through clinical trials that one plant-derived lactic acid bacteria (LAB) can improve constipation. We preliminarily found that the plant-derived LAB Lactococcus lactis BM32-1 can grow in a mixture of sericin and fibroin, which are extracted from silk and have been reported to help promote health. Thus, in the present study, we evaluated the favorable effect of a sericin/fibroin mixture (S/F-M), which was extracted from silk prepared from cocoons reared in an aseptic rearing system using an artificial diet, fermented with the BM32-1 strain through a clinical trial. The trial was conducted at Hiroshima University from June to October 2022 as a double-blind, placebo-controlled, randomized parallel-group comparative study with 50 eligible subjects (aged 23-71) who had an average defecation frequency of less than 5 times per week. The subjects were instructed to drink 100 mL of fermented S/F-M or placebo every day. After the 12 weeks of the clinical trial period, the average defecation frequency increased significantly-1.4 times higher than that at baseline in the test group-as compared with the placebo group. Furthermore, the fecal microbiota was also compared before and after treatment, revealing that intake of the fermented S/F-M significantly multiplied the relative abundance of the genera Enterococcus and Clostridium, which have been reported to contribute to the amelioration of constipation by improving the gut microbiota and producing butyric acid, respectively. In conclusion, the S/F-M fermented using the BM32-1 strain improves defecation frequency through alteration of the gut microbiota.

Bioengineered Silk Protein-Based 3D In Vitro Models for Tissue Engineering and Drug Development: From Silk Matrix Properties to Biomedical Applications.

3D in vitro model has emerged as a valuable tool for studying tissue development, drug screening, and disease modeling. 3D systems can accurately replicate tissue microstructures and physiological features, mirroring the in vivo microenvironment departing from conventional 2D cell cultures. Various 3D in vitro models utilizing biomacromolecules like collagen and synthetic polymers have been developed to meet diverse research needs and address the complex challenges of contemporary research. Silk proteins, bearing structural and functional similarities to collagen, have been increasingly employed to construct advanced 3D in vitro systems, surpassing the limitations of 2D cultures. This review examines silk proteins' composition, structure, properties, and functions, elucidating their role in 3D in vitro models. Furthermore, recent advances in biomedical applications involving silk-based organoid models are discussed. In particular, the unique physiological attributes of silk matrix constituents in in vitro tissue constructs are highlighted, providing a meticulous evaluation of their importance. Additionally, it outlines the current research hurdles and complexities while contemplating future avenues, thereby paving the way for developing complex and biomimetic silk protein-based microtissues.

Enhancing angiogenesis in peri-implant soft tissue with bioactive silk fibroin microgroove coatings on zirconia surfaces.

Zirconia abutments and restorations have improved the aesthetic appeal of implant restoration, yet peri-implantitis poses a significant threat to long-term success. The soft tissue surrounding implants is a crucial biological barrier against inflammation and subsequent bone loss. Peri-implantitis, akin to periodontitis, progresses rapidly and causes extensive tissue damage. Variations in tissue structure significantly influence disease progression, particularly the lower vascular density in peri-implant connective tissue, compromising its ability to combat infection and provide essential nutrients. Blood vessels within this tissue are vital for healing, with angiogenesis playing a key role in immune defense and tissue repair. Enhancing peri-implant soft tissue angiogenesis holds promise for tissue integration and inflammation control. Microgroove surfaces have shown potential in guiding vessel growth, but using subtractive technologies to carve microgrooves on zirconia surfaces may compromise mechanical integrity. In this study, we utilized inkjet printing to prepare bioactive silk fibroin microgrooves (SFMG) coating with different sizes on zirconia surfaces. SFMG coating, particularly with 90 µm width and 10 µm depth, effectively directed human umbilical vein endothelial cells (HUVECs) along microgrooves, promoting their proliferation, migration, and tube formation. The expression of vascular endothelial growth factor A and fibroblast growth factor in HUVECs growing on SFMG coating was upregulated. Additionally, the SFMG coating activated the PI3K-AKT pathway and increased glycolytic enzyme gene expression in HUVECs. In conclusion, SFMG coating enhances HUVEC growth and angiogenesis potential by activating the PI3K-AKT pathway and glycolysis, showing promise for improving tissue integration and mitigating inflammation in zirconia abutments and restorations.

Unveiling the synergy: Biocompatible alginate-cellulose hydrogel loaded with silk fibroin and zinc ferrite nanoparticles for enhanced cell adhesion, and anti-biofilm activity.

Combining a biocompatible hydrogel scaffold with the cell-supportive properties of silk fibroin (SF) and the unique functionalities of ZnFe2O4 nanoparticles creates a promising platform for advanced nanobiomaterials. The research is centered on synthesizing a natural hydrogel using cellulose (Cellul) and sodium alginate (SA) combined with SF and zinc ferrite nanoparticles. A range of analytical and biological assays were conducted to determine the biological and physicochemical properties of the nanobiocomposite. The hemolysis and 2,5-diphenyl-2H-tetrazolium bromide (MTT) assays indicated that the SA-Cellul hydrogel/SF/ZnFe2O4 nanobiocomposite was a biocompatible against human dermal fibroblasts (Hu02) and red blood cells (RBC). In addition, aside from demonstrating outstanding anti-biofilm activity, the nanobiocomposite also promotes the Hu02 cells adhesion, showcasing the synergistic effect of incorporating SF and ZnFe2O4 nanoparticle. These promising results show that this nanobiocomposite has potential applications in various biomedical fields.

Evaluating bio-physicochemical properties of raw powder prepared from whole larvae containing liquid silk of the domestic silkworm.

The domestic silkworm, Bombyx mori, has been widely used in silk production for centuries. It is also used as a bioreactor by the textile and pharmaceutical industries to mass produce recombinant bioactive proteins containing silk-based materials. Furthermore, silkworms are well-known as a source of food and have also been orally administered to prevent and treat several human disorders. In this study, we aimed to investigate the inherent bio-physicochemical properties of edible silkworms to accurately evaluate their clinical and nutritional potential. We prepared raw powder from whole larvae of silkworm. The yield rate of the powder derived from dried larvae was almost 100% (98.1-99.1% in replicates). As "percentage yield" translates to "Budomari" in Japanese, this raw powder was named "B100rw." We further prepared B100dn that was denatured through autoclaving. Thereafter, we examined whether B100rw sustained the original bio-physicochemical properties by comparing it with B100dn. There was no significant difference in nutritional content between B100rw and B100dn. B100rw contained proteins derived from silkworm larvae and mulberry leaves, whereas the proteins of B100dn were mostly degraded. On measuring the enzymatic activity of both powders using trehalase as an indicator enzyme, B100rw was found to maintain trehalase activity. B100rw also maintained a random coil conformation, similar to that of liquid silk. This suggested that B100rw sustained the unique bio-physicochemical properties of living larvae. These findings may facilitate the development of novel food products or orally administered vaccines.

A hybrid structure based on silk fibroin/PVA nanofibers and alginate/gum tragacanth hydrogel embedded with cardamom extract.

Hybrid structures made of natural-synthetic polymers have been interested due to high biological features combining promising physical-mechanical properties. In this research, a hybrid dressing consisting of a silk fibroin (SF)/polyvinyl alcohol (PVA) nanofibers and sodium alginate (SA)/gum tragacanth (GT) hydrogel incorporating cardamom extract as an antibacterial agent was prepared. Accordingly, SF was extracted from cocoons followed by electrospinning in blend form with PVA (SF/PVA ratio: 1:1) under the voltage of 18 kV and the distances of 15 cm. The SEM images confirmed the formation of uniform, bead free fibers with the average diameter of 199 ± 28 nm. FTIR and XRD results revealed the successful extraction of SF and preparation of mixed fibrous mats. Next, cardamom oil extract-loaded SA/GT hydrogel was prepared and the nanofibrous structure was placed on the surface of hydrogel. SEM analysis depicted the uniform morphology of hybrid structure with desirable matching between two layers. TGA analysis showed desired thermal stability. The swelling ratio was found to be 1251% after 24 h for the hybrid structure and the drug was released without any initial burst. MTT assay and cell attachment results showed favorable biocompatibility and cell proliferation on samples containing extract, and antibacterial activity values of 85.35% against S. aureus and 75% against E. coli were obtained as well. The results showed that the engineered hybrid nanofibrous-hydrogel film structure incorporating cardamom oil extract could be a promising candidate for wound healing applications and skin tissue engineering.

Silk fibroin catheter with stable bioinspired inner-surfaces for inhibition of bioadhesion.

Biofilm formation on indwelling medical devices such as catheters and ventilators due to the adhesion of bacteria poses significant challenges in healthcare. Surface modification with micro- and nano-structures offers a promising strategy to prevent bioadhesion and is safer than surface chemical modification approaches. Here, catheters were prepared using silk fibroin (SF) hydrogels and an infusion molding method, with the inner surface featuring a micropapillae structure inspired by lotus leaves (SF-CMP). After phenylethanol (PEA) fumigation treatment, the resulting catheters (SF-CMP PEA) displayed improved swelling resistance and mechanical properties compared to methanol-treated catheters (SF-CMP MeOH). PEA was more efficient than methanol in controlling the size, distribution, and content of silk crystalline ß-sheet blocks and thus the swelling and mechanical properties. Moreover, the micro-papillae structure on SF-CMP PEA remained stable over 35 days in solution, in contrast to SF-CMP MeOH, which lasted <7 days. SF-CMP PEA exhibited repellent effects against E. coli and S. aureusin vitro, and low cytotoxicity to the endothelial cells cultured on the unpatterned surface. Additionally, subcutaneous implantation studies showed reduced inflammation around the micropatterned samples compared to controls with a plain, unpatterned surface. The unique properties of SF-based materials, including tunable structures, biocompatibility, degradation, and drug-loading capability make them an attractive material for anti-bioadhesion in applications ranging from indwelling medical devices to tissue engineering scaffolds.

Preparation of Autoclavable and Injectable Silk Fibroin Cryogels for Tissue Engineering Applications.

A cryogel is a supermacroporous gel network that is generated at subzero temperatures by polymerizing monomers or gelating polymeric precursors. Since cryogels possess inherent characteristics such as interconnected macroporous structures, excellent mechanical properties, and high resistance to autoclave sterilization, they are highly desirable for tissue engineering and regenerative medicine. Silk fibroin, a natural protein obtained from Bombyx mori silkworms, is an excellent raw material for cryogel preparation. The aim of this study is to establish a controlled method for preparing silk fibroin cryogels with suitable properties for application as tissue engineering scaffolds. Using a dual crosslinking strategy consisting of low-temperature radical polymerization coupled with methanol-induced conformational transformation, porous cryogels are prepared. The cryogels display many unique characteristics, such as an interconnected macroporous structure, a high water absorption capacity, water-triggered shape memory, syringe injectability, and strong resilience to autoclave sterilization. Furthermore, the cryogels demonstrate excellent biocompatibility and cell affinity, facilitating cell adhesion, migration, and proliferation. The interconnected supermacroporous architecture resembling the native extracellular matrix, together with their unique physical properties and autoclaving stability, suggests that cryogels are promising candidate scaffolds for tissue engineering and cell therapy.

Study on the Structure and Properties of Silk Fibers Obtained from Factory All-Age Artificial Diets.

The traditional production mode of the sericulture industry is no longer suitable for the development requirements of modern agriculture; to facilitate the sustainable development of the sericulture industry, factory all-age artificial diet feeding came into being. Understanding the structural characteristics and properties of silk fibers obtained from factory all-age artificial diet feeding is an important prerequisite for application in the fields of textiles, clothing, biomedicine, and others. However, there have been no reports so far. In this paper, by feeding silkworms with factory all-age artificial diets (AD group) and mulberry leaves (ML group), silk fibers were obtained via two different feeding methods. The structure, mechanical properties, hygroscopic properties, and degradation properties were studied by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). Structurally, no new functional groups appeared in the AD group. Compared with the ML group, the structure of the two groups was similar, and there was no significant difference in mechanical properties and moisture absorption. The structure of degummed silk fibers is dominated by crystalline regions, but α-chymotrypsin hydrolyzes the amorphous regions of silk proteins, so that after 28 d of degradation, the weight loss of both is very small. This provides further justification for the feasibility of factory all-age artificial diets for silkworms.

Tailoring silk fibroin fibrous architecture by a high-yield electrospinning method for fast wound healing possibilities.

In this study, a novel array electrospinning collector was devised to generate two distinct regenerated silk fibroin (SF) fibrous membranes: ordered and disordered. Leveraging electrostatic forces during the electrospinning process allowed precise control over the orientation of SF fiber, resulting in the creation of membranes comprising both aligned and randomly arranged fiber layers. This innovative approach resulted in the development of large-area membranes featuring exceptional stability due to their alternating patterned structure, achievable through expansion using the collector, and improving the aligned fiber membrane mechanical properties. The study delved into exploring the potential of these membranes in augmenting wound healing efficiency. Conducting in vitro toxicity assays with adipose tissue-derived mesenchymal stem cells (AD-MSCs) and normal human dermal fibroblasts (NHDFs) confirmed the biocompatibility of the SF membranes. We use dual perspectives on exploring the effects of different conditioned mediums produced by cells and structural cues of materials on NHDFs migration. The nanofibers providing the microenvironment can directly guide NHDFs migration and also affect the AD-MSCs and NHDFs paracrine effects, which can improve the chemotaxis of NHDFs migration. The ordered membrane, in particular, exhibited pronounced effectiveness in guiding directional cell migration. This research underscores the revelation that customizable microenvironments facilitated by SF membranes optimize the paracrine products of mesenchymal stem cells and offer valuable physical cues, presenting novel prospects for enhancing wound healing efficiency.