Nanofiber Scaffolds Research Articles

Design of cellulose nanocrystal‐integrated poly(vinyl alcohol)/polyethylene glycol electrospun nanofiber scaffolds for biomedical applications

AbstractThe present study aims to fabricate cellulose nanocrystals (CNCs) doped poly(vinyl alcohol) polyethylene glycol (PVA/PEG) nanocomposite films for biomedical applications. The nanocomposite films were prepared by using electrospinning techniques with varying percentages of CNCs (10% and 20%) mixed with PVA and PEG (50:50). The properties of the produced nanocomposites were evaluated using a battery of tests. The results of surface morphology and structural miscibility obtained from field emission‐scanning electron microscopy (FE‐SEM), atomic force microscopy, and Fourier transformer infra‐red studies revealed appreciable compatibility among the three components (CNCs, PVA, and PEG). FE‐SEM micrographs revealed that the nanomaterials underwent a dramatic transition from discontinuous to continuous fibers, with interconnected or network‐like fibers. The constructed scaffold material's stability went up from three to four phases, according to thermal and degradation experiments. In addition, research on biodegradation and water absorption found that CNCs outperformed pure PVA and PVA/PEG in terms of swelling percentage time and enzyme degradation rate. The hemolysis assay of the PVA/PEG/CNC scaffold showed good compatibility (below 5%) and according to the MTT assay, both NIH 3T3 and L929 cells survived well, indicating that the CNCs could serve as a useful scaffold for tissue engineering applications such skin tissue regeneration. In sum, the findings proved that biological domains including tissue engineering and wound healing can benefit from produced nanofiber scaffolds.

Decellularized Extracellular Matrix Scaffolds: Recent Advances and Emerging Strategies in Bone Tissue Engineering.

Bone tissue engineering (BTE) is a complex biological process involving the repair of bone tissue with proper neuronal network and vasculature as well as bone surrounding soft tissue. Synthetic biomaterials used for BTE should be biocompatible, support bone tissue regeneration, and eventually be degraded in situ and replaced with the newly generated bone tissue. Recently, various forms of bone graft materials such as hydrogel, nanofiber scaffolds, and 3D printed composite scaffolds have been developed for BTE application. Decellularized extracellular matrix (DECM), a kind of natural biological material obtained from specific tissues and organs, has certain advantages over synthetic and exogenous biomaterial-derived bone grafts. Moreover, DECM can be developed from a wide range of biological sources and possesses strong molding abilities, natural 3D structures, and bioactive factors. Although DECM has shown robust osteogenic, proangiogenic, immunomodulatory, and bone defect healing potential, the rapid degradation and limited mechanical properties should be improved for bench-to-bed translation in BTE. This review summarizes the recent advances in DECM-based BTE and discusses emerging strategies of DECM-based BTE.

Alginate-gelatin based nanocomposite hydrogel scaffold incorporated with bioactive glass nanoparticles and fragmented nanofibers promote osteogenesis: From design to in vitro studies

The current study proposes fragmented nanofibers of polycaprolactone (FNF) with bioactive glass nanoparticles (nBG) incorporated into a polymeric matrix of alginate-gelatin for the creation of a hydrogel scaffold. Four groups were prepared: control, bioactive glass containing scaffold (BG), fragmented nanofibers with bioactive glass scaffold (FNF(PCL) + BG), and fragmented composite nanofibers scaffold (FNF (PCL + BG)). FNF (PCL + BG) scaffolds revealed a more controlled degradation rate, with approximately 20 % degradation occurring after 28 compared. The FNF(PCL) + BG scaffolds had the highest compressive strength in both dry and wet states. Following 14 days of incubation in simulated body fluid, hydroxyapatite formation had occurred on the surface of scaffolds containing nBG, and after 28 days on other groups tested. Cell studies revealed that the FNF(PCL) + BG scaffolds had superior cell viability without inhibiting cell proliferation. The FNF(PCL) + BG and FNF(PCL + BG) scaffolds had the highest alkaline phosphatase (ALP) activity and FNF(PCL) + BG scaffolds showed to support osteogenic differentiation.

The Impact of Helium and Nitrogen Plasmas on Electrospun Gelatin Nanofiber Scaffolds for Skin Tissue Engineering Applications

The Impact of Helium and Nitrogen Plasmas on Electrospun Gelatin Nanofiber Scaffolds for Skin Tissue Engineering Applications

Electrospun nanofibrous scaffolds reinforced with therapeutic lithium/manganese-doped calcium phosphates: Advancing skin cancer therapy through apoptosis induction

In the current study we fabricated potent materials by incorporating therapeutic elements into calcium phosphates (CPs) to combat cancer. This involved synthesizing manganese (Mn)- and lithium (Li)-doped CPs and loading them into electrospun nanofibers (NFs) composed of chitosan (CS) and polyethylene oxide (PEO). The characterized CPs exhibited excellent properties, including a particle size of 47–75 nm, surface charge of –(30−56) mV, and specific surface area of 75–266 m2/g. The electrochemical analysis revealed that Mn and Mn/Li-doped CPs are promising for generating oxygen free radicals and H2O2, crucial for cancer therapy. Biological evaluation showcased the outstanding performance of the developed materials. MTT assay revealed a cytotoxic effect of nano-constructs on melanoma A375 cell line without adverse effects on normal L929 cells over 72 h. Annexin V/PI apoptosis assay indicated substantial apoptosis rates in A375 cells treated with PC-20 % (62.55 ± 4.59 %). The obtained data of qPCR analysis of pro-apoptotic and anti-apoptotic genes (P53, Bax, Bcl-2) in A375 cells treated with different CP nanoparticles (NPs) showed a significant increase in P53 and Bax gene expression, indicating high levels of A375 cell apoptosis. Additionally, the samples containing Mn ion exhibited high reactive oxygen species (ROS) generation. In conclusion, the fabricated NFs scaffolds hold promising potential for cancer therapy.

One-Step Gas Foaming Strategy for Constructing Strontium Nanoparticle Decorated 3D Scaffolds: a New Platform for Repairing Critical Bone Defects.

The management of critical-sized bone defects poses significant clinical challenges, particularly in the battlefield and trauma-related injuries. However, bone tissue engineering scaffolds that satisfy high porosity and good angiogenic and osteogenic functions are scarce. In this study, 3D nanofiber scaffolds decorated with strontium nanoparticles (3DS-Sr) were fabricated by combining electrospinning and gas foaming. Sodium borohydride (NaBH4) served a dual role as both a reducing and gas-foaming agent, enabling a one-step process for expansion and modification. In vitro experimental results demonstrated that 3DS-Sr possessed an integrated multilayered porous structure. It promoted angiogenesis by upregulating the expression of hypoxia-inducible factor-1α (HIF-1α) protein and phosphorylation of ERK through the sustained release of Sr2+ and created a favorable microenvironment for osteogenesis by activating the Wnt/β-catenin pathway. In vivo experiments indicated that 3DS-Sr promoted cranial bone regeneration by synergistically promoting the effects of vascularization and osteogenesis. In summary, this study proposed a bioactive bone scaffold in a "one stone, two birds" manner, providing a promising strategy for bone defect repair.

Pullulan/Collagen Scaffolds Promote Chronic Wound Healing via Mesenchymal Stem Cells

This study investigated the development of Pullulan/Collagen nanofiber scaffolds integrated with mesenchymal stem cells (MSCs) to enhance chronic wound healing. The combination of these biopolymers aims to optimize the scaffold properties for cell growth, viability, and tissue regeneration. Materials and Methods: Pullulan, Collagen, and Pullulan/Collagen composite nanofibers were fabricated using electrospinning. The fibers were characterized using scanning electron microscopy (SEM) to determine the fiber diameter, and Fourier-transform infrared spectroscopy (FTIR) was employed to assess the molecular interactions. Cell viability was evaluated using MSCs cultured on the scaffolds and apoptosis assays were conducted to assess cell health. Distilled water was used as the solvent to maximize biocompatibility. Results: SEM analysis revealed that Pullulan nanofibers exhibited a larger average diameter (274 ± 20 nm) compared to Collagen fibers (167.03 ± 40.04 nm), while the Pullulan/Collagen composite fibers averaged 280 ± 102 nm. FTIR confirmed the molecular interactions between Pullulan and Collagen. Regarding biocompatibility, the Pullulan/Collagen scaffold demonstrated superior cell viability at 99% compared to 91% for Pullulan alone. Apoptosis assays indicated significantly lower necrosis rates for the composite scaffold (1.29%) than for the Pullulan-only scaffolds (2.35%). Conclusion: The use of distilled water as a solvent played a critical role in increasing cell viability and facilitating healthy proliferation of MSCs without cellular damage. Additionally, the reduced platelet activation and macrophage activity (0.75-fold for both) further supported the biocompatibility of the Pullulan/Collagen scaffold, demonstrating its potential for tissue engineering and chronic wound healing applications.

Electrospinning Aligned SF/Magnetic Nanoparticles-Blend Nanofiber Scaffolds for Inducing Skeletal Myoblast Alignment and Differentiation.

In the realm of skeletal muscle tissue engineering, anisotropic materials that emulate natural tissues show substantial promise. Electrospun scaffolds, mimicking the fibrillar structure of the extracellular matrix, are commonly employed but often fall short in achieving optimal alignment and mechanical strength. Silk fibroin has emerged as a versatile material in tissue engineering, valued for its biocompatibility, mechanical robustness, and biodegradability. However, conventional electrospinning methods of SF result in randomly oriented fibers, limiting their efficacy. In this work, we developed a straightforward method to fabricate directional tissue scaffolds using silk fibroin. By integrating a magnetic field collecting device and incorporating Fe3O4 nanoparticles into the spinning solution, we successfully produced well-aligned silk nanofiber scaffolds. These aligned fibers not only improved scaffold orientation and mechanical properties but also exhibited magnetic responsiveness. The aligned SF scaffolds effectively guided the adhesion, proliferation, and differentiation of mesenchymal stem cells along the fiber direction. Cultured on these scaffolds, myoblast C2C12 cells demonstrated oriented growth, highlighting the potential of aligned SF fibers in advancing skeletal muscle engineering for biomedical applications.

Electrospun Pt-TiO2 nanofibers Doped with HPA for Catalytic Hydrodeoxygenation

Electrospinning is utilized to fabricate catalytic nanofiber scaffold for biocrude upgrading in hydrodeoxygenation (HDO) following computational studies suggesting the need for nano-catalysts for efficient HDO conversion and selectivity. Here, Pt-TiO2 nanofibers are fabricated through electrospinning, followed by wet impregnation with a heteropoly acid (HPA), tungstosilicic acid. Intensive heat treatments were incorporated during and after processes to obtain a HPA doped Pt-TiO2 nano-catalyst. Catalytic HDO was performed in a batch reactor with phenol as the raw biocrude dissolved in hexadecane. The HPA doped Pt-TiO2 catalyst demonstrated promising HDO performance of 37.2% conversion and a 78.9% selectivity to oxygen free benzene and the remainder 21.1% as diphenyl ester as a result of esterification by acidic components of the catalyst. Additionally, BET surface area characterization show a low surface area 16.9 m2 g−1 significantly lower than existing commercial catalysts and a mesoporous nature suitable for selectivity. The presence of HPA on the anatase nanofiber compensated for low platinum nanoparticles crystallinity on the nanofibers. This work might create needed alternatives for preparing HDO catalysts for efficient aromatics production.

3D Nanofiber-Assisted Embedded Extrusion Bioprinting for Oriented Cardiac Tissue Fabrication.

Three-dimensional (3D) bioprinting technology stands out as a promising tissue manufacturing process to control the geometry precisely with cell-loaded bioinks. However, the isotropic culture environment within the bioink and the lack of topographical cues impede the formation of oriented cardiac tissue. To overcome this limitation, we present a novel method named 3D nanofiber-assisted embedded bioprinting (3D-NFEP) to fabricate cardiac tissue with an oriented morphology. Aligned 3D nanofiber scaffolds were fabricated by divergence electrospinning, which provided structural support for printing of the low-viscosity bioink and structural induction to cardiomyocytes. Cells adhered to the aligned fibers after hydrogel degradation, and a high degree of cell alignment was observed. This technology was also demonstrated as a feasible solution for multilayer cell printing. Therefore, 3D-NFEP was demonstrated as a promising method for bioprinting oriented cardiac tissue with low-viscosity bioink and is expected to be applied for structured and cardiac tissue engineering.

Bio-Templated Aerogel Fibers: Heterogeneous Spinodal Architecting and In Situ Fibrillation of Thermoplastic Polyurethane-Silica on Nanostructured Cellulose Nanofiber Scaffold for Enhanced Thermomechanical Performance.

This study addresses the inherent fragility and fractal limitations of traditional silica aerogels by developing a bio-templated aerogel fiber. Integrating cellulose nanofibers (CNFs), thermoplastic polyurethane (TPU), and silica aerogel (SA) in a dimethyl sulfoxide (DMSO) dispersion, a gel-spinning technique was employed to create aerogel fibers with superior thermomechanical performance. CNF also provided excellent rheological modification for successful spinnability, fast gelation, and fiber formation. The unique hierarchical structure of these fibers, formed through hot-stretching and surface modification, combined the superior mechanical strength and flexibility of TPU with the exceptional insulation properties of CNF and SA. The CNF network, encapsulated within the SA particles, formed a core-shell structure, axially aligned, that significantly enhances the thermal stability and mechanical performance of the fibers while maintaining a lightweight and porous architecture. Comprehensive morphological, thermal, and mechanical analyses were conducted to evaluate the properties of the developed aerogel fibers. Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy verified the successful surface modification and grafting reactions on CNF, contributing to improved hydrophobicity and thermal insulation. Adjusting the CNF content allowed for tailoring of the thermomechanical characteristics, with a notable 294% increase in tensile strength from 5.3 to 15.6 MPa and an enhanced crystallization temperature from 106 to 119.97 °C. Furthermore, cyclic tensile and compression tests validated the durability and shape recovery capabilities of the aerogel fibers, making them promising candidates for high-performance applications in extreme environments. The thermal conductivity validated by experimental data further highlights the potential of CNF-based aerogel fibers as sustainable and multifunctional materials for advanced thermal insulation, mechanical reinforcement, and flexible structural applications.

Electrospun polyvinyl alcohol nanofiber scaffolds incorporated strontium-substituted hydroxyapatite from sand lobster shells: synthesis, characterization, and in vitro biological properties

The paper describes the synthesis of hydroxyapatite (HAp) and strontium-substituted hydroxyapatite (SrHAp) from sand lobster shells by a hydrothermal method. The HAp and SrHAp were incorporated into the polyvinyl alcohol (PVA) nanofiber scaffold through the eletrospinning method. The scaffolds were incorporated with 5wt% of hydroxyapatite (HAp), 5wt%, 10wt%, and 15% of SrHAp. The physicochemical, mechanical, and in vitro biological properties of the scaffold were evaluated. The incorporation of HAp or SrHAp was evidenced by the diffraction patterns and the phosphate functional groups related to HAp. The morphological results showed the decrement of fiber diameter in line with the increased SrHAp concentration. A tensile test was conducted to investigate the mechanical properties of the scaffolds, and the results showed that the scaffolds perform poorly at a higher SrHAp concentration because of exceeding agglomeration levels. The PVA/SrHAp15 performed the best antibacterial activity against E. coli and S. aureus with an inhibition zone of (15.2 ± 0.2) and (14.5 ± 0.8), respectively. The apatite formation was more abundant in PVA/SrHAp10 after immersion in a simulated body fluid (SBF). Cell viability results showed that the scaffold enabled the osteoblast cells to grow and proliferate. The biocompatibility of HAp and SrHAp resulted in the enhancement of cell adhesion. Based on all tests, the PVA/SrHAp 10 scaffold shows a strong candidate for further in vivo studies.

Investigating the impact of curcumin extract incorporation on PVA/Collagen/Chitosan/HAp nanofiber scaffold: morphological, mechanical, wettability, degradation rate, and antibacterial activity

This study investigates the impact of curcumin extract incorporation on the properties of PVA/Collagen/Chitosan/HAp nanofiber scaffolds for bone tissue engineering applications. Nanofibers were fabricated via electrospinning using PVA, collagen, chitosan, hydroxyapatite (HAp), and curcumin. Their morphology, mechanical properties, wettability, degradation rate, and antibacterial activity were comprehensively evaluated. Fourier transform infrared (FTIR) spectroscopy confirmed the successful incorporation of all components into the nanofibers and the potential for interactions between their functional groups. Scanning Electron Microscopy (SEM) revealed bead-free, fine nano-fibers with a decrease in diameter ranging from 139 nm to 108 nm. Simultaneously, enhanced mechanical properties were observed, with ultimate strength increasing from 2.8 MPa to 8.8 MPa as the diameter decreased. Notably, 15% curcumin extract maintained favorable hydrophilicity and degradation rates, crucial factors for tissue regeneration. Antibacterial activity was also enhanced, with an inhibition zone of 6.71 mm against E.coli observed at 15% curcumin treatment. Staphylococcus aureus exhibited the largest zone of inhibition (8.74 mm) at 15% curcumin concentration. This research demonstrates the feasibility of incorporating curcumin into nanofiber scaffolds for bone tissue engineering applications.

LDH nanoparticles-doped cellulose nanofiber scaffolds with aligned microchannels direct high-efficiency neural regeneration and organized neural circuit remodeling through RhoA/Rock/Myosin II pathway

Spinal cord injury (SCI) triggers interconnected malignant pathological cascades culminating in structural abnormalities and composition changes of neural tissues and impairs spinal cord tissue function. Cellulose nanofibers (CNF) have considerable potential in mimicking tissue microstructure for nerve regeneration, but the effectiveness of CNF in repairing SCI remains poorly understood. In this study, we designed a Mg–Fe layered double hydroxide (LDH)-doped cellulose nanofiber (CNF) scaffold with aligned intact microchannels and homogeneously distributed pores (CNF-LDH), loaded with retinoic acid and sonic hedgehog (CNF-LDH-RS) for neuroregeneration. The aligned microchannel structure and chemical cues in the scaffold were designed further to enhance the differentiation of neural stem cells towards neurons and promote axon growth while inhibiting differentiation to astrocytes. Transplanting the scaffolds into a completely transected SCI mice model dramatically improved behavioral and electrophysiological outcomes underpinned by robust neuronal regeneration, significant axonal growth and orderly neural circuit remodeling. RNA-seq analysis revealed the pivotal roles of the RhoA/Rock/Myosin II pathway and neuroactive ligand-receptor interaction pathway in SCI repair by CNF-LDH-RS. Particularly, Myosin II emerged as a key gene for functional recovery, and its effect on negative regulation of axon growth was suppressed by the scaffolds, resulting in a distinctly oriented growth of the axons along the microchannel structure. The results indicate that CNF-LDH scaffolds rationally combined with physical and biochemical cues create promising tissue-engineered substrates to facilitate the repair of spinal cord injury.

Ultralight 3D silk nanofiber scaffolds with enhanced hemostatic properties and biocompatibility for rapid hemostasis

Developing an effective hemostatic material that combines excellent biocompatibility with high blood absorption capacity remains a critical clinical challenge. In this study, ultralight three-dimensional (3D) silk fibroin (SF) scaffolds, composed of continuously interconnected nanofibers, are introduced as a novel hemostatic material. The distinctive fluffy nanofiber architecture endows the scaffolds with a remarkable porosity of 78.9 % and exceptional liquid absorption capacity. The integration of montmorillonite (MMT) further enhances the hemostatic properties of the scaffolds, while SF naturally promotes the intrinsic coagulation pathway. The scaffolds exhibit an impressive blood absorption capacity of 1687.7 %. In vitro evaluations indicate that the SF nanofiber scaffolds exhibit a blood clotting index rate of 24.6 %, a hemolysis ratio of less than 4 %, and outstanding biocompatibility. The blood coagulation time is significantly reduced from 449 s to 230 s. Additionally, the scaffolds show adhesion rates to erythrocytes and platelets of 52 % and 45.9 %, respectively. The nanofiber scaffolds aggregate and activate platelets extensively, accelerating the formation of platelet emboli, which collectively contribute to their superior hemostatic performance. Consequently, these nanofiber scaffolds hold substantial promise as absorbable hemostatic agents for achieving rapid hemostasis in clinical applications.

The nanocellulose fiber scaffold to construct a hierarchical phenolic coating on cotton fiber surfaces for inactivating pathogens through an enhanced protein adsorption mechanism

Emerging pathogens present a significant societal threat, and biological protection textiles are expected to play a pivotal role in controlling their spread. However, incorporating highly effective pathogen transmission-blocking abilities into textiles while ensuring their large-scale production remains challenging. This work has successfully developed a hierarchically structured coating for cotton fibers, which exhibits enhanced antiviral and antibacterial functionalities compared to existing phenolic coating methods. The multilevel coating consisted of a cellulose nanofibers (CNFs) scaffold on cotton fibers, along with a phenolic layer constructed of 4,4-bis(4-hydroxyphenyl)valeric acid (DPA) on the CNF surfaces. The bioactive textile exhibited remarkable antiviral and antibacterial capabilities with exceptional durability, while demonstrating strong protein affinity for pathogen destruction. Especially, the CNFs with diameters below 10 nm show significant size effect in capturing phi6. This is primarily due to their complementary shape matching the spike proteins, resulting in an increased number of binding sites and improved inactivation effectiveness. Moreover, the modification process was demonstrated to be scalable in a pad-dry-cure line commonly employed by textile finishing factories, and the resultant coating ensured reliable safety for human skin without sacrificing wearing comfort. This approach opens up new possibilities for developing protective textiles that effectively block pathogen transmission.

Cell migration within porous electrospun nanofibrous scaffolds in a mouse subcuticular implantation model.

Cellular infiltration into electrospun nanofibers (NFs) is limited due to the dense structure and small pore sizes. We developed a programmed NF collector that can fabricate porous NFs with desired pore sizes and thickness. Previously we demonstrated improved cellular proliferation and differentiation of osteoblasts, osteoclasts, and fibroblasts with increased pore sizes of polycaprolactone (PCL) NF in-vitro. This study investigated in-vivo host cell migration and vascular ingrowth within porous NF sheets implanted subcutaneously in a mouse model. Two types of PCL NFs with well-defined pore sizes were created using varying speeds of the NF collector: NF-zero (no movement, pore size 14.4 ± 8.9 µm2) and NF-high (0.232 mm/min, pore size 286.7 ± 381.9 µm2). The NF obtained by using classical flat NF collector (2D NF, pore size 1.09 ± 1.7 µm2) was a control. The three formulae of NFs were implanted subcutaneously in 18 BALB/cJ mice. Animals were killed 7 and 28-days after implantation (n = 3 per group per time point). The tissue with implanted NFs were collected for histologic analysis. Overall, 7-day samples showed little inflammatory response. At 28-days, the degree of tissue penetration of PCL NF sheet matrices was linked to pore size and area. NFs with the largest pore area had more efficient tissue migration and new blood vessel formation compared to those with smaller pore sizes. No newly formed blood vessels were observed in the 2D NF group. A porous NF scaffold with controllable pore size has potential for tissue repair/regeneration in situ with potential for many applications in orthopaedics.

Advanced alginate-based nanofiber aerogels: A synthetic matrix for high-efficiency lysozyme adsorption and controlled release

The development of materials with high lysozyme adsorption is critical for drug delivery and skin wound applications, as it enhances antibacterial properties, stability, and controlled release of therapeutic agents, thereby improving treatment efficacy and safety. Alginate-based nanofiber scaffolds, featuring high surface area and multiple adsorption sites, can efficiently absorb lysozyme and regulate its release through tunable pore channels, offering a promising approach to chronic wound management. In this study, we fabricated poly (vinyl alcohol-co-ethylene) (EVOH) nanofiber-based sodium alginate (ENSA) aerogels using a simple two-step crosslinking procedure. The resulting aerogels, with controllable porosity formed via high-pressure spraying techniques (aerogel film) and molding (aerogel sponge), were evaluated for their high-loading capacity and controllable release of lysozyme. The aerogel film exhibited a remarkable lysozyme adsorption capacity of 1965 ± 36 mg/g, while the aerogel sponge sustained lysozyme release over 14 days. Analysis of the drug-release mechanism through four kinetic models revealed two distinct processes: cation exchange and matrix diffusion. The aerogel's pore structure influenced the diffusion processes, enabling tailored drug release profiles. Additionally, the ENSA aerogels demonstrated good mechanical properties, non-cytotoxicity, and potent antibacterial activity, positioning them as promising materials for skin wound dressings.

Bioactive stem cell-laden 3D nanofibrous scaffolds for tissue engineering

This study presents biomimetic nanoscaffolds composed of electrospun polycaprolactone-collagen (PCL-Coll) nanofibers, loaded with bioactive Arnebia euchroma (AE) extract and stem cells, to develop cell-based tissue engineering constructs. The incorporation of AE extract, known for its antioxidant and anti-inflammatory properties, into the PCL-Coll nanofibers resulted in nanoscaffolds denoted as PCL-Coll/AE0, PCL-Coll/AE5, PCL-Coll/AE10, and PCL-Coll/AE15, corresponding to AE extract concentrations of 0.0, 5.0, 10.0, and 15.0 wt%, respectively. The PCL-Coll/AE nanoscaffolds exhibited high porosity values of 62.81 ± 4.78 %, 59.9 ± 2.10 %, 52.44 ± 2.66 %, and 46.32 ± 1.35 %, respectively, thereby providing an optimal 3D environment for cell attachment and proliferation. Analysis of the AE extract revealed the presence of abundant shikonin, a key bioactive compound, as indicated by its characteristic absorption bands at 490, 520, and 560 nm. FE-SEM data confirmed the homogeneous immobilization of AE compounds within the 3D nanofiber scaffolds, with fibers averaging 316 ± 137 nm in diameter, falling within the range of natural collagen fibers. To evaluate the structural integrity of the fully stem cell-laden scaffolds, we assessed cell growth, cell attachment within the PCL-Coll/AE nanoscaffolds, the cytoprotective effects of AE extract, and the expression levels of stemness-related genes, including Nanog, Rex1, Sox2, Oct4, Klf4, and C-Myc. Real-time PCR assays indicated that key indicators of stemness were upregulated, with average increases from 120 ± 15 % in PCL-Coll/AE0 to 250 ± 30 % in PCL-Coll/AE15 over a two-week period. These upregulations promote cell proliferation and facilitate cell cycle progression. Data indicate that increasing the concentration of bioactive AE extract within the scaffolds enhanced cell adhesion on the scaffold surface and improved cell viability after 7 days of culture, with viability rising from 109 ± 6.15 % in PCL-Coll/AE0 to 145.5 ± 10.11 % in PCL-Coll/AE15. Cytoprotective assays against oxidative stress induced by H₂O₂ revealed relative cell viability for PCL-Coll/AE0, PCL-Coll/AE5, PCL-Coll/AE10, and PCL-Coll/AE15 as 42.78 ± 5.85 %, 49.29 ± 6.79 %, 64.98 ± 3.32 %, and 78.68 ± 3.09 %, respectively. These results correlate with the antioxidant potency of AE extract compounds, particularly shikonin and its derivatives. Therefore, the cell-laden biomimetic PCL-Coll/AE15 scaffolds, which demonstrate sustained stemness features and a continuous local release of bioactive AE compounds, represent a promising candidate for tissue engineering applications.

Construction of Thick Myocardial Tissue through Layered Seeding in Multi-Layer Nanofiber Scaffolds.

A major challenge in myocardial tissue engineering is replicating the heart's highly complex three-dimensional (3D) anisotropic structure. Heart-on-a-chip (HOC) is an emerging technology for constructing myocardial tissue in vitro in recent years, but most existing HOC systems face difficulties in constructing 3D myocardial tissue aligned with multiple cell layers. Electrospun nanofibers are commonly used as scaffolds for cell growth in myocardial tissue engineering, which can structurally simulate the extracellular matrix to induce the aligned growth of myocardial cells. Here, we developed an HOC that integrates multi-layered aligned polycaprolactone (PCL) nanofiber scaffolds inside microfluidic chips, and constructed 3D thick and aligned tissue with a layered seeding approach. By culturing human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) on chip, the myocardial tissue on the two layered nanofibers reached a thickness of ~53 μm compared with ~19 μm for single-layered nanofibers. The obtained myocardial tissue presented well-aligned structures, with densely distributed α-actinin. By the third day post seeding, the hiPSC-CMs contract highly synchronously, with a contraction frequency of 18 times/min. The HOC with multi-layered biomimetic scaffolds provided a dynamic in vitro culture environment for hiPSC-CMs. Together with the layered cell-seeding process, the designed HOC promoted the formation of thick, well-aligned myocardial tissue.