Physiological cyclic stretching potentiates the cell-cell junctions in vascular endothelial layer formed on aligned fiber substrate.

In vascular tissue engineering, formation of stable endothelial cell-cell and cell-substrate adhesions is essential for maintaining long-term patency of the tissue-engineered vascular grafts (TEVGs). In this study, sheet-like aligned fibrous substrates of poly(l-lactide-co-caprolactone) (PLCL) were prepared by electrospinning to provide basement membrane-resembling structural support to endothelial cells (ECs). Cyclic stretching at physiological and pathological levels was then applied to human umbilical vein endothelial cells (HUVECs) cultured on chosen fibrous substrate using a force-loading device, from which effects of the cyclic stretching on cell-cell and cell-substrate adhesions were examined. It was found that applying uniaxial 1 Hz cyclic stretch at physiological levels (5 % and 10 % elongation) strengthened the cell-cell junctions, thus leading to improved structural integrity, functional expression and resistance to thrombin-induced damaging impacts in the formed endothelial layer. The cell-cell junctions were disrupted at pathological level (15 % elongation) cyclic stretching, which however facilitated the formation of focal adhesions (FAs) at cell-substrate interface. Mechanistically, the effects of cyclic stretching on endothelial cell-cell and cell-substrate adhesions were identified to be correlated with the RhoA/ROCK signaling pathway. Results from this study highlight the relevance between applying dynamic mechanical stimulation and maintaining the structural integrity of the formed endothelial layer, and implicate a necessity to implement appropriate dynamic mechanical training (i.e., preconditioning) to obtain tissue-engineered blood vessels with long-term patency post-implantation.

A hydrogel-based first-aid tissue adhesive with effective hemostasis and anti-bacteria for trauma emergency management.

BACKGROUND: Clinical tissue adhesives remain some critical drawbacks for managing emergency injuries, such as inadequate adhesive strength and insufficient anti-infection ability. Herein, a novel, self-healing, and antibacterial carboxymethyl chitosan/polyaldehyde dextran (CMCS/PD) hydrogel is designed as the first-aid tissue adhesive for effective trauma emergency management. METHODS: We examined the gel-forming time, porosity, self-healing, antibacterial properties, cytotoxicity, adhesive strength, and hemocompatibility. Liver hemorrhage, tail severance, and skin wound infection models of rats are constructed in vivo, respectively. RESULTS: Results demonstrate that the CMCS/PD hydrogel has the rapid gel-forming (~ 5 s), good self-healing, and effective antibacterial abilities, and could adhere to tissue firmly (adhesive strength of ~ 10 kPa and burst pressure of 327.5 mmHg) with excellent hemocompatibility and cytocompatibility. This suggests the great prospect of CMCS/PD hydrogel in acting as a first-aid tissue adhesive for trauma emergency management. The CMCS/PD hydrogel is observed to not only achieve rapid hemostasis for curing liver hemorrhage and tail severance in comparison to commercial hemostatic gel (Surgiflo ®) but also exhibit superior anti-infection for treating acute skin trauma compared with clinical disinfectant gel (Prontosan ®). CONCLUSIONS: Overall, the CMCS/PD hydrogel offers a promising candidate for first-aid tissue adhesives to manage the trauma emergency. Because of the rapid gel-forming time, it could also be applied as a liquid first-aid bandage for mini-invasive surgical treatment.

Carbon dots induce pathological damage to the intestine via causing intestinal flora dysbiosis and intestinal inflammation.

BACKGROUND: Carbon dots (CDs), as excellent antibacterial nanomaterials, have gained great attention in treating infection-induced diseases such as periodontitis and stomatitis. Given the eventual exposure of CDs to the intestine, elucidating the effect of CDs on intestinal health is required for the safety evaluation of CDs. RESULTS: Herein, CDs extracted from ε-poly-L-lysine (PL) were chosen to explore the modulation effect of CDs on probiotic behavior in vitro and intestinal remodeling in vivo. Results verify that PL-CDs negatively regulate Lactobacillus rhamnosus (L. rhamnosus) growth via increasing reactive oxygen species (ROS) production and reducing the antioxidant activity, which subsequently destroys membrane permeability and integrity. PL-CDs are also inclined to inhibit cell viability and accelerate cell apoptosis. In vivo, the gavage of PL-CDs is verified to induce inflammatory infiltration and barrier damage in mice. Moreover, PL-CDs are found to increase the Firmicutes to Bacteroidota (F/B) ratio and the relative abundance of Lachnospiraceae while decreasing that of Muribaculaceae. CONCLUSION: Overall, these evidences indicate that PL-CDs may inevitably result in intestinal flora dysbiosis via inhibiting probiotic growth and simultaneously activating intestinal inflammation, thus causing pathological damage to the intestine, which provides an effective and insightful reference for the potential risk of CDs from the perspective of intestinal remodeling.

In situ electrospun aloe-nanofiber membrane for chronic wound healing.

Alleviating excessive inflammation while accelerating chronic wound healing to prevent wound infection has remained challenging, especially during the coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 when patients experienced difficulties with receive appropriate healthcare. We addressed this issue by developing handheld electrospun aloe-nanofiber membranes (ANFMs) with convenient, environmentally friendly properties and a therapeutic capacity for wound closure. Our results showed that ANFMs fabricated with high molecular weight polyvinyl alcohol (PVA) to form fibers during electrospinning had uniform fibrous architecture and a porous structure. Given the value of aloe gel in accelerating wound healing, liquid extracts from ANFMs significantly downregulated the expression of the pro-inflammatory genes, interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), and markedly suppress the generation of reactive oxygen species (ROS) induced by lipopolysaccharide in RAW264.7 macrophages. These results indicated the excellent antioxidant and anti-inflammatory effects of ANFMs. After implantation into a mouse diabetic wound model for 12 days in situ, ANFMs notably expedited chronic wound healing via promoting angiogenesis and enhancing cell viability. Our ANFMs generated by handheld electrospinning in situ healed chronic wounds offer a convenient and promising alternative for patients to heal their own wounds under variable conditions.

Step-wise CAG@PLys@PDA-Cu2+ modification on micropatterned nanofibers for programmed endothelial healing.

Native-like endothelium regeneration is a prerequisite for material-guided small-diameter vascular regeneration. In this study, a novel strategy is proposed to achieve phase-adjusted endothelial healing by step-wise modification of parallel-microgroove-patterned (i.e., micropatterned) nanofibers with polydopamine-copper ion (PDA-Cu2+) complexes, polylysine (PLys) molecules, and Cys-Ala-Gly (CAG) peptides (CAG@PLys@PDA-Cu2+). Using electrospun poly(l-lactide-co-caprolactone) random nanofibers as the demonstrating biomaterial, step-wise modification of CAG@PLys@PDA-Cu2+ significantly enhanced substrate wettability and protein adsorption, exhibited an excellent antithrombotic surface and outstanding phase-adjusted capacity of endothelium regeneration involving cell adhesion, endothelial monolayer formation, and the regenerated endothelium maturation. Upon in vivo implantation for segmental replacement of rabbit carotid arteries, CAG@PLys@PDA-Cu2+ modified grafts (2 mm inner diameter) with micropatterns on inner surface effectively accelerated native-like endothelium regeneration within 1 week, with less platelet aggregates and inflammatory response compared to those on non-modified grafts. Prolonged observations at 6- and 12-weeks post-implantation demonstrated a positive vascular remodeling with almost fully covered endothelium and mature smooth muscle layer in the modified vascular grafts, accompanied with well-organized extracellular matrix. By contrast, non-modified vascular grafts induced a disorganized tissue formation with a high risk of thrombogenesis. In summary, step-wise modification of CAG@PLys@PDA-Cu2+ on micropatterned nanofibers can significantly promote endothelial healing without inflicting thrombosis, thus confirming a novel strategy for developing functional vascular grafts or other blood-contacting materials/devices.

Eugenol eliminates carbapenem-resistant Klebsiella pneumoniae via reactive oxygen species mechanism.

Multidrug-resistant (MDR) bacterial infections have gained increasing attention due to the high incidence rates and high mortality, especially for the carbapenem-resistant Klebsiella pneumoniae (CRKP) infection that can cause severe complications (e.g., pneumonia and sepsis) in multiple organs. Therefore, the development of new antibacterial agents against CRKP is imperative. Inspired by natural plant antibacterial agents with broad-spectrum antibacterial properties, the antibacterial/biofilm activity of eugenol (EG) on CRKP and their underlying mechanisms are investigated in our work. It is found that EG exhibits remarkable inhibitory effects on planktonic CRKP in a dose-dependent fashion. Meanwhile, the destruction of membrane integrity induced by the formation of reactive oxygen species (ROS) and glutathione reduction results in the leakage of bacterial cytoplasmic components, including DNA, ß-galactosidase, and protein. Moreover, when EG contacts with bacterial biofilm, the whole thickness of the dense biofilm matrix decreases, and the integrity is destroyed. Overall, this work verified that EG could eliminate CRKP via ROS-induced membrane rupture, which offers vital evidence to explain the antibacterial ability of EG against CRKP.

Recent Progress in antibacterial hydrogel coatings for targeting biofilm to prevent orthopedic implant-associated infections.

The application of orthopedic implants for bone tissue reconstruction and functional restoration is crucial for patients with severe bone fractures and defects. However, the abiotic nature of orthopedic implants allows bacterial adhesion and colonization, leading to the formation of bacterial biofilms on the implant surface. This can result in implant failure and severe complications such as osteomyelitis and septic arthritis. The emergence of antibiotic-resistant bacteria and the limited efficacy of drugs against biofilms have increased the risk of orthopedic implant-associated infections (OIAI), necessitating the development of alternative therapeutics. In this regard, antibacterial hydrogels based on bacteria repelling, contact killing, drug delivery, or external assistance strategies have been extensively investigated for coating orthopedic implants through surface modification, offering a promising approach to target biofilm formation and prevent OIAI. This review provides an overview of recent advancements in the application of antibacterial hydrogel coatings for preventing OIAI by targeting biofilm formation. The topics covered include: (1) the mechanisms underlying OIAI occurrence and the role of biofilms in exacerbating OIAI development; (2) current strategies to impart anti-biofilm properties to hydrogel coatings and the mechanisms involved in treating OIAI. This article aims to summarize the progress in antibacterial hydrogel coatings for OIAI prevention, providing valuable insights and facilitating the development of prognostic markers for the design of effective antibacterial orthopedic implants.

Engineering a Mechanoactive Fibrous Substrate with Enhanced Efficiency in Regulating Stem Cell Tenodifferentiation.

Electrospun-aligned fibers in ultrathin fineness have previously demonstrated a limited capacity in driving stem cells to differentiate into tendon-like cells. In view of the tendon's mechanoactive nature, endowing such aligned fibrous structure with mechanoactivity to exert in situ mechanical stimulus by itself, namely, without any forces externally applied, is likely to potentiate its efficiency of tenogenic induction. To test this hypothesis, in this study, a shape-memory-capable poly(l-lactide-co-caprolactone) (PLCL) copolymer was electrospun into aligned fibrous form followed by a "stretching-recovery" shape-programming procedure to impart shape memory capability. Thereafter, in the absence of tenogenic supplements, human adipose-derived stem cells (ADSCs) were cultured on the programmed fibrous substrates for a duration of 7 days, and the effects of constrained recovery resultant stress-stiffening on cell morphology, proliferation, and tenogenic differentiation were examined. The results indicate that the in situ enacted mechanical stimulus due to shape memory effect (SME) did not have adverse influence on cell viability and proliferation, but significantly promoted cellular elongation along the direction of fiber alignment. Moreover, it revealed that tendon-specific protein markers such as tenomodulin (TNMD) and tenascin-C (TNC) and gene expression of scleraxis (SCX), TNMD, TNC, and collagen I (COL I) were significantly upregulated on the mechanoactive fibrous substrate with higher recovery stress compared to the counterparts. Mechanistically, the Rho/ROCK signaling pathway was identified to be involved in the substrate self-actuation-induced enhancement in tenodifferentiation. Together, these results suggest that constrained shape recovery stress may be employed as an innovative loading modality to regulate the stem cell tenodifferentiation by presenting the fibrous substrate with an aligned tendon-like topographical cue and an additional mechanoactivity. This newly demonstrated paradigm in modulating stem cell tenodifferentiation may improve the efficacy of tendon tissue engineering strategy for tendon healing and regeneration.

Synergistic effects of mechanical stimulation and crimped topography to stimulate natural collagen development for tendon engineering.

Suitable scaffold structures and mechanical loading are essential for functional tendon engineering. However, the bipolar fibril structure of native tendon collagen is yet to be recaptured in engineered tendons. This study compared the development of Achilles tendons of postnatal rats with and without (via surgical section) mechanical loading to define the mechanism of mechanical stimulation-mediated tendon development. The results demonstrated that the severed tendons weakened mechanically and exhibited disorganization without a bipolar fibril superstructure. Proteomic analysis revealed differentially expressed key regulatory molecules related to the collagen assembly process, including decreased fibromodulin, keratocan, fibroblast growth factor-1, and increased lumican and collagen5a1 in the severed tendons with immunohistochemical verification. Additionally, a complex regulatory network of mechanical stimulation-mediated collagen assembly in a spatiotemporal manner was also revealed using bioinformatics analysis, wherein PI3K-Akt and HDAC4 may be the predominant signaling pathways. A wavy microgrooved surface (Y = 5.47sin(0.015x)) that biomimics tendon topography was observed to enhance the expression of collagen assembly molecules under mechanical loading, and the aforementioned pathways are particularly involved and verified with their respective inhibitors of LY-294002 and LMK-235. Furthermore, an electrospun crimped nanofiber scaffold (approximately 2 µm fiber diameter and 0.12 crimpness) was fabricated to biomimic the tenogenic niche environment; this was observed to be more effective on enhancing collagen production and assembly under mechanical stimulation. In conclusion, the synergistic effect between topographical niche and mechanical stimulation was observed to be essential for collagen assembly and maturation and should be applied to functional tendon engineering in the future. STATEMENT OF SIGNIFICANCE: In biomaterial-mediated tendon regeneration, mechanical stimulation is essential for tendon collagen assembly. However, the underlying mechanisms remain not fully defined, leading to the failure of the native-like collagen regeneration. In this study, a mechanical stimulation deprivation model of rat tendon was established to reveal the mechanisms in tendon development and define the key regulatory molecules including small leucine-rich proteoglycans, lysyl oxidase and collagen V. After ensuring the importance of biomimetic structure in tendon remodeling, crimped nanofibers were developed to verify these regulatory molecules, and demonstrated that mechanical stimulation significantly enhanced collagen assembly via PIK3 and HDAC4 pathways in biomaterial-regulated tendon regeneration. This study provides more insightful perspectives in the physiologically remodeling progression of tendon collagen and design of tendon scaffolds.

Comparative Study of Traditional Single-Needle Electrospinning and Novel Spiral-Vane Electrospinning: Influence on the Properties of Poly(caprolactone)/Gelatin Nanofiber Membranes.

Spiral-vane electrospinning (SVE), a novel needleless electrospinning, was proven effective in obtaining high-throughput production of nanofibers. However, the properties of the electrospun nanofibers produced by SVE remain relatively underexplored, especially in comparison with those made by traditional single-needle electrospinning (SNE). Hence, for the comparative study of SNE and SVE in this study, the difference in the preparation mechanism was first analyzed using numerical simulation, followed by the experimental analysis of the effects of spinneret types on the quality and biocompatibility of electrospun poly(caprolactone)/gelatin (PCL/Gel) nanofibers. The values predicted by the electric field results were consistent with the experimental data, showing that the PCL/Gel nanofibers prepared by SVE have higher yields than SNE. Although the different spinnerets (i.e., needle and spiral vane) had little effect on the surface chemistry, thermal stability, and composition of the PCL/Gel nanofibers, they had great effects on the fiber diameter distribution and mechanical properties in which SVE-electrospun nanofibers have the wider diameter distribution and higher softness. Furthermore, the SVE-electrospun nanofibers were also proven to exhibit good biocompatibility for cell growth of human adipose-derived stem cells (hADSCs) and cell-fiber interactions. Summarily, compared to the traditional SNE, SVE-electrospun nanofibers exhibited many merits including high-throughput yield, good air permeability, and compliance, which provide a facile and effective platform for the improvement of nanofiber applications in biomedical fields (e.g., tissue engineering, cosmetic, and medical textiles).

An overview of substrate stiffness guided cellular response and its applications in tissue regeneration.

Cell-matrix interactions play a critical role in tissue repair and regeneration. With gradual uncovering of substrate mechanical characteristics that can affect cell-matrix interactions, much progress has been made to unravel substrate stiffness-mediated cellular response as well as its underlying mechanisms. Yet, as a part of cell-matrix interaction biology, this field remains in its infancy, and the detailed molecular mechanisms are still elusive regarding scaffold-modulated tissue regeneration. This review provides an overview of recent progress in the area of the substrate stiffness-mediated cellular responses, including 1) the physical determination of substrate stiffness on cell fate and tissue development; 2) the current exploited approaches to manipulate the stiffness of scaffolds; 3) the progress of recent researches to reveal the role of substrate stiffness in cellular responses in some representative tissue-engineered regeneration varying from stiff tissue to soft tissue. This article aims to provide an up-to-date overview of cell mechanobiology research in substrate stiffness mediated cellular response and tissue regeneration with insightful information to facilitate interdisciplinary knowledge transfer and enable the establishment of prognostic markers for the design of suitable biomaterials.

Engineering a Highly Biomimetic Chitosan-Based Cartilage Scaffold by Using Short Fibers and a Cartilage-Decellularized Matrix.

Engineering scaffolds with structurally and biochemically biomimicking cues is essential for the success of tissue-engineered cartilage. Chitosan (CS)-based scaffolds have been widely used for cartilage regeneration due to its chemostructural similarity to the glycosaminoglycans (GAGs) found in the extracellular matrix of cartilage. However, the weak mechanical properties and inadequate chondroinduction capacity of CS give rise to compromised efficacy of cartilage regeneration. In this study, we incorporated short fiber segments, processed from electrospun aligned poly(lactic-co-glycolic acid) (PLGA) fiber arrays, into a citric acid-modified chitosan (CC) hydrogel scaffold for mechanical strengthening and structural biomimicking and meanwhile introduced cartilage-decellularized matrix (CDM) for biochemical signaling to promote the chondroinduction activity. We found that the incorporation of PLGA short fibers and CDM remarkably strengthened the mechanical properties of the CC hydrogel (+349% in compressive strength and +153% in Young's modulus), which also exhibited a large pore size, appropriate porosity, and fast water absorption ability. Biologically, the engineered CDM-Fib/CC scaffold significantly promoted the adhesion and proliferation of chondrocytes and supported the formation of matured cartilage tissue with a cartilagelike structure and deposition of abundant cartilage ECM-specific GAGs and type II collagen (+42% in GAGs content and +295% in type II collagen content). The enhanced mechanical competency and chondroinduction capacity with the engineered CDM-Fib/CC scaffold eventually fulfilled successful in situ osteochondral regeneration in a rabbit model. This study thereby demonstrated a great potential of the engineered highly biomimetic chitosan-based scaffold in cartilage tissue repair and regeneration.

Regeneration of Subcutaneous Cartilage in a Swine Model Using Autologous Auricular Chondrocytes and Electrospun Nanofiber Membranes Under Conditions of Varying Gelatin/PCL Ratios.

The scarcity of ideal biocompatible scaffolds makes the regeneration of cartilage in the subcutaneous environment of large animals difficult. We have previously reported the successful regeneration of good-quality cartilage in a nude mouse model using the electrospun gelatin/polycaprolactone (GT/PCL) nanofiber membranes. The GT/PCL ratios were varied to generate different sets of membranes to conduct the experiments. However, it is unknown whether these GT/PCL membranes can support the process of cartilage regeneration in an immunocompetent large animal model. We seeded swine auricular chondrocytes onto different GT/PCL nanofiber membranes (GT:PCL = 30:70, 50:50, and 70:30) under the sandwich cell-seeding mode. Prior to subcutaneously implanting the samples into an autologous host, they were cultured in vitro over a period of 2 weeks. The results revealed that the nanofiber membranes with different GT/PCL ratios could support the process of subcutaneous cartilage regeneration in an autologous swine model. The maximum extent of homogeneity in the cartilage tissues was achieved when the G5P5 (GT: PC = 50: 50) group was used for the regeneration of cartilage. The formed homogeneous cartilage tissues were characterized by the maximum cartilage formation ratio. The extents of the ingrowth of the fibrous tissues realized and the extents of infiltration of inflammatory cells achieved were found to be the minimum in this case. Quantitative analyses were conducted to determine the wet weight, cartilage-specific extracellular matrix content, and Young's modulus. The results indicated that the optimal extent of cartilage formation was observed in the G5P5 group. These results indicated that the GT/PCL nanofiber membranes could serve as a potential scaffold for supporting subcutaneous cartilage regeneration under clinical settings. An optimum GT/PCL ratio can promote cartilage formation.

Shape Memory and Osteogenesis Capabilities of the Electrospun Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Modified Poly(l-Lactide) Fibrous Mats.

Poly(l-lactide) (PLLA) as one of the most well-known biodegradable polyesters has been studied extensively for bone tissue engineering. If being properly programmed, scaffolds from PLLA can also be endowed with the capability of shape memory. However, several noted issues, for example, mechanical brittleness, high glass transition temperature Tg, and relatively poor shape retention and recovery properties, necessitate modification of the PLLA to improve its application efficacy in physiological conditions. This study is proposed to modify PLLA by having the biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) incorporated to form ultrafine composite fibers (i.e., PLLA-PHBV) through electrospinning. Different pairs of PLLA-PHBV at the varying mass ratios of 10:0, 9:1, 8:2, 7:3, 6:4, and 0:10 can be successfully electrospun into fibrous form with the fineness of 2-3 µm. Incorporation of PHBV enables to give rise to desired Tg decreases and also, interestingly, increases in the Young's modulus of the PLLA-PHBV blends, while gradually increasing the PHBV mass ratios up to 30%. The PLLA-PHBV (7:3) formulation is identified to present excellent shape memory properties with high shape fixing ratio (>98%) and shape recovery ratio (>96%) compared to the unmodified PLLA fiber counterpart. Moreover, the PLLA-PHBV (7:3) fibers also show enhanced osteogenesis-inducing ability in the mouse bone mesenchymal stem cells, even under nonosteoinductive conditions. Collectively, for the first time this study demonstrates the enhanced shape memory and osteogenesis capabilities of the electrospun PLLA-PHBV composite fibers, and the researched PLLA-PHBV (7:3) fiber system could be potentially applied as a multifunctional scaffolding material for applications in bone tissue repair and regeneration. Impact statement By first converting the poly(l-lactide) (PLLA)-poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) hybrids into fibrous form at varied mass ratios followed by a thorough characterization, we reasonably demonstrated that incorporation of an appropriate amount of PHBV (i.e., 30%) into the PLLA fibers could give rise to significant improvement on the shape memory capability of the PLLA, along with the desired decreases in the transition temperature (Tg). Moreover, the fibrous PLLA-PHBV (7:3) scaffold was also found to significantly promote the osteogenic commitment in bone mesenchymal stem cells with osteoinductive factors in a synergistic manner. Our biomimicking and shape memory enabled fibrous scaffold of PLLA-PHBV could be used to construct multifunctional three-dimensional scaffold with shape memory effect for bone regeneration.

Understanding the cellular responses based on low-density electrospun fiber networks.

Fibers produced from electrospinning are well-known to be extremely fine with diameters ranging from tens of nanometers to a few microns. Such ultrafine fibers not only allow for engineering scaffolds resembling the ultrastructure of the native extracellular matrix, but also offer possibility to explore the remodeling behavior of cells in vitro, due to their mechanically 'adequate' softness endowed by their ultrafine fineness. However, the remodeling effect of cells on the biomimicking fibrous substrates remains to be understood, because the crisscrossing and entangling among nanofibers in those tightly packed fibrous mats ultimately lead to merely a topological phenomenon, similar to that of the nanofiber-like topography embossed on the surface of a solid matter. In this study, the effect of nanofiber density on cellular response behavior was investigated by reducing the density of electrospun fiber networks. Using polycaprolactone (PCL) as a model polymer, randomly oriented fiber networks with various densities, namely, 37.7 ± 16.3 µg/cm2 (D1), 103.8 ± 16.3 µg/cm2 (D2), 198.2 ± 40.0 µg/cm2 (D3), and 471.8 ± 32.7 µg/cm2 (D4), were prepared by electrospinning for varied collection durations (10 s, 50 s, 100 s, and 10 min, respectively). By examining the responsive behavior of the human induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSCs) cultured on these nanofibrous networks, we showed that the fiber network with a moderate density (D2) is beneficial to the cell attachment, spreading, actin polymerization, contractility and migration. There also showed an increased tendency in nuclear localization of the Yes-associated protein (YAP) and subsequent activation of YAP responsive gene transcription, and cell proliferation and collagen synthesis were also enhanced on the D2. However, further increasing the fiber density (D3, D4) gave rise to weakened induction effect of fibers on the cellular responses. These results enrich our understanding on the effect of fiber density on cell behavior, and disclose the dependence of cellular responses on fiber density. This study paves the way to precisely design biomimetic fibrous scaffolds for achieving enhanced cell-scaffold interactions and tissue regeneration.

Stiffness of the aligned fibers affects structural and functional integrity of the oriented endothelial cells.

Promoting healthy endothelialization of the tissue-engineered vascular grafts is of great importance in preventing the occurrence of undesired post-implantation complications including neointimal hyperplasia, late thrombosis, and neoatherosclerosis. Previous researches have demonstrated the crucial role of scaffold topography or stiffness in modulating the behavior of the monolayer endothelial cells (ECs). However, effects of the stiffness of scaffolds with anisotropic topography on ECs within vivo like oriented morphology has received little attention. In this study, aligned fibrous substrates (AFSs) with tunable stiffness (14.68-2141.72 MPa), similar to the range of stiffness of the healthy and diseased subendothelial matrix, were used to investigate the effects of fiber stiffness on ECs' attachment, orientation, proliferation, function, remodeling and dysfunction. The results demonstrate that stiffness of the AFSs, capable of providing topographical cues, is a crucial endothelium-protective microenvironmental factor by maintaining stable and quiescent endothelium with in vivo like orientation and strong cell-cell junctions. Stiffer AFSs exacerbated the disruption of endothelium integrity, the occurrence of endothelial-to-mesenchymal transition (EndMT), and the inflammation-induced activation in the endothelial monolayer. This study provides new insights into the understanding on how the stiffness of biomimicking anisotropic substrate regulates the structural and functional integrity of the in vivo like endothelial monolayer, and offers essential designing parameters in engineering biomimicking small-diameter vascular grafts for the regeneration of viable blood vessels. STATEMENT OF SIGNIFICANCE: In vascular tissue engineering, promoting endothelialization on scaffold surface has been considered as a paramount strategy to reduce post-implantation complications. Electrospun aligned fibers have been known to provide contact guidance effect in directing endothelial cells' oriented growth, however, whether the formed EC monolayer in 'correct' orientation shape is of 'correct' function hasn't been explored yet. Given the recognized important role of substrate stiffness in endothelial function, AFSs across physiologically relevant range of moduli (14.68-2141.72 MPa) while maintaining consistent surface chemistry and topographical features were employed to investigate the fiber stiffness effects on ECs function in anisotropic morphology. This study will provide more insightful perspectives in the physiologically remodeling progression of vascular endothelium and design of vascular scaffolds.

Electrospun acid-neutralizing fibers for the amelioration of inflammatory response.

Biodegradable aliphatic polyesters, especially polylactide (PLA), polyglycolide (PGA), and their copolymer poly(lactide-co-glycolide) (PLGA), are the most representative and widely used synthetic polymers in the field of tissue engineering and regenerative medicine. However, these polyesters often give rise to aseptic inflammation because of their acidic degradation products after implantation. Here, unidirectional shell-core structured fibers of chitosan/poly(lactide-co-glycolide) (i.e., CTS/PLGA) with acid-neutralizing capability were developed for addressing the noted issue by coating the PLGA fiber surfaces with a layer of the alkaline chitosan by coaxial electrospinning. Our results showed that during a period of 8-week degradation, the shell-layer of chitosan with its unique alkaline nature for acid-neutralization obviously hindered the pH decrease as a result of the degradation of PLGA-core. In a mocked acidic environment testing of the human dermal fibroblasts, chitosan-enabled acidity neutralization could significantly reduce in vitro the secretion of inflammatory factors and downregulate the expression of related inflammatory genes. Thereafter, biocompatibility assessment in vitro showed that the CTS/PLGA fibers had poorer cell adhesion capacity than the PLGA fibers but were cytocompatible and promoted cell migration and secretion of collagen. Moreover, subcutaneous embedding for two and four weeks in vivo revealed that the CTS/PLGA fibers significantly reduced the recruitment of inflammatory cells and the formation of foreign body giant cells (FBGCs). This study thereby demonstrated the evident acid-neutralizing effect of the chitosan-coating layer on alleviating the inflammatory responses caused by the acidic degradation products of the PLGA-core. Our highly aligned CTS/PLGA fibers, as a kind of quasi "pH-neutral fibers" with the acid-neutralizing capability, could be potentially applied for engineering those architecturally anisotropic tissues (e.g., tendon/ligament) toward improved efficacy of regeneration. STATEMENT OF SIGNIFICANCE: It is well known that acidic degradation products from representative aliphatic polyesters (e.g., PLA, PGA, and PLGA) give rise to the problem of aseptic inflammation. Various alkaline components acting as neutralizing agents have been used to address the noted issue. However, rather less attention has been paid to engineer these polyesters into a fibrous form with acid-neutralizing functionality. The present study proposes the concept of "pH-neutral fibers" and develops shell-core structured unidirectional fibers of chitosan/poly(lactide-co-glycolide) with acid-neutralizing capability for ameliorating inflammatory responses caused by the acidic degradation products of PLGA. It provides a comprehensive study encompassing fiber characterization and in vitro and in vivo evaluation, which would pave the way for developing sophisticated pH-neutral fibers for functional tissue regeneration.

Stiffness of Aligned Fibers Regulates the Phenotypic Expression of Vascular Smooth Muscle Cells.

Electrospun uniaxially aligned ultrafine fibers show great promise in constructing vascular grafts mimicking the anisotropic architecture of native blood vessels. However, understanding how the stiffness of aligned fibers would impose influences on the functionality of vascular cells has yet to be explored. The present study aimed to explore the stiffness effects of electrospun aligned fibrous substrates (AFSs) on phenotypic modulation in vascular smooth muscle cells (SMCs). A stable jet coaxial electrospinning (SJCES) method was employed to generate highly aligned ultrafine fibers of poly(l-lactide- co-caprolactone)/poly(l-lactic acid) (PLCL/PLLA) in shell-core configuration with a remarkably varying stiffness region from 0.09 to 13.18 N/mm. We found that increasing AFS stiffness had no significant influence on the cellular shape and orientation along the fiber direction with the cultured human umbilical artery SMCs (huaSMCs) but inhibited the cell adhesion rate, promoted cell proliferation and migration, and especially enhanced the F-actin fiber assembly in the huaSMCs. Notably, higher fiber stiffness resulted in significant downregulation of contractile markers like alpha-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain, calponin, and desmin, whereas upregulated the gene expression of pathosis-associated osteopontin ( OPN) in the huaSMCs. These results allude to the phenotype of huaSMCs on stiffer AFSs being miserably modulated into a proliferative and pathological state. Consequently, it adversely affected the proliferation and migration behavior of human umbilical vein endothelial cells as well. Moreover, stiffer AFSs also revealed to incur significant upregulation of inflammatory gene expression, such as interleukin-6 ( IL-6), monocyte chemoattractant protein-1 ( MCP-1), and intercellular adhesion molecule-1 ( ICAM-1), in the huaSMCs. This study stresses that although electrospun aligned fibers are capable of modulating native-like oriented cell morphology and even desired phenotype realization or transition, they might not always direct cells into correct functionality. The integrated fiber stiffness underlying is thereby a critical parameter to consider in engineering structurally anisotropic tissue-engineered vascular grafts to ultimately achieve long-term patency.

Fabrication of high performance silk fibroin fibers via stable jet electrospinning for potential use in anisotropic tissue regeneration.

Regenerated silk fibroin (SF) from Bombyx mori silkworm cocoons is a highly regarded natural protein-biomaterial suitable for engineering a variety of biological tissues. Electrospinning offers a unique approach to fiber formation that can readily produce micro- and nano-scale fibers recapitulating the ultrastructure of a native extracellular matrix. However, SF fibers from conventional electrospinning suffer from the problem of poor mechanical properties for load-bearing relevant tissue regeneration applications. In this study, highly aligned high-strength SF fibers were fabricated by a recently emerged stable jet electrospinning (SJES) approach, with the aid of high molecular weight poly(ethylene oxide) (PEO) acting as a fiber-forming ingredient to increase control over the jetting instability during electrospinning. The results showed that 90% of the collected SF/PEO (mass ratio 88 : 12) fiber assembly via SJES oriented unidirectionally with an angle variation of <1° and displayed obvious anisotropic wettability. Mechanically, the as-electrospun highly aligned SF/PEO fibers exhibited a 22.0-fold increase in ultimate tensile strength (50.85 ± 1.13 MPa) and a 49.3-fold increase in Young's modulus (1185.99 ± 164.56 MPa) compared with the randomly oriented SF fibers. A subsequent methanol treatment further remarkably boosted the tensile strength to 73.91 ± 5.15 MPa and Young's modulus to 2426.13 ± 86.67 MPa. The mechanical performance of the SF fibers via SJES was also impressive, even when tested in the wet state. The substantial improvement in the mechanical properties of the electrospun SF fibers is attributed to the SJES-enabled higher molecular orientation and contents of the secondary structure (α-helix and ß-pleated sheet), as well as the high degree of fiber alignment. Moreover, biological tests verified that these SF-based fibrous scaffolds supported the induced pluripotent stem cell derived mesenchymal stem cells to adhere, migrate and grow in a manner of orienting along the fiber axis. We speculate that these high-performance biomimicking SF fibers might give rise to improved efficacy while being utilized to architecturally regenerate anisotropic load-bearing tissues (e.g., tendon, ligament, and blood vessel).

Highly aligned core-shell structured nanofibers for promoting phenotypic expression of vSMCs for vascular regeneration.

This study was designed to assess the efficacy of hyaluronan (HA) functionalized well-aligned nanofibers of poly-l-lactic acid (PLLA) in modulating the phenotypic expression of vascular smooth muscle cells (vSMCs) for blood vessel regeneration. Highly aligned HA/PLLA nanofibers in core-shell structure were prepared using a novel stable jet electrospinning approach. Formation of a thin HA-coating layer atop each PLLA nanofiber surface endowed the uni-directionally oriented fibrous mats with increased anisotropic wettability and mechanical compliance. The HA/PLLA nanofibers significantly promoted vSMC to elongation, orientation, and proliferation, and also up-regulated the expression of contractile genes/proteins (e.g., α-SMA, SM-MHC) as well as the synthesis of elastin. Six weeks of in vivo scaffold replacement of rabbit carotid arteries showed that vascular conduits made of circumferentially aligned HA/PLLA nanofibers could maintain patency and promoted oriented vSMC regeneration, lumen endothelialization, and capillary formation. This study demonstrated the synergistic effects of nanotopographical and biochemical cues in one biomimetic scaffold design for efficacious vascular regeneration.