Electrospun nanofiber mesh with connective tissue growth factor and mesenchymal stem cells for pelvic floor repair: Long-term study.

Pelvic organ prolapse (POP) affects many women, with an estimated lifetime risk of surgical intervention of 18.7%. There is a need for alternative approaches as the use of synthetic nondegradable mesh was stopped due to severe adverse events, and as current methods for pelvic floor repair have high POP recurrence rates. Thus, we hypothesized that electrospun degradable meshes with stem cells and growth factor were safe and durable for the long term in elderly rats. In an abdominal repair model, electrospun polycaprolactone (PCL) meshes coated with connective tissue growth factor (CTGF)/PEG-fibrinogen (PF) and rat mesenchymal stem cells were implanted in elderly female rats and removed after in average 53 weeks (53-week group). Collagen amount and production were quantified by qPCR and Western blotting. Moreover, histological appearance and biomechanical properties were evaluated. Results were compared with previous results of young rats with identical mesh implanted for 24 weeks (24-week group). The 53-week group differed from the 24-week group in terms of (1) reduced collagen III, (2) strong reduction in foreign body response, and (3) altered histological appearance. We found comparable biomechanical properties, aside from higher, not significant, mean tissue stiffness in the 53-week group. Lastly, we identified mesh components 53 weeks after implantation. This study provides new insights into future POP repair in postmenopausal women by showing how CTGF/PF-coated electrospun PCL meshes with stem cells exhibit sufficient support, biocompatibility, and no mesh-related complications long term in an abdominal repair model in elderly rats.

Real-Time Measurement of Cell Mechanics as a Clinically Relevant Readout of an In Vitro Lung Fibrosis Model Established on a Bioinspired Basement Membrane.

Lung fibrosis, one of the major post-COVID complications, is a progressive and ultimately fatal disease without a cure. Here, an organ- and disease-specific in vitro mini-lung fibrosis model equipped with noninvasive real-time monitoring of cell mechanics is introduced as a functional readout. To establish an intricate multiculture model under physiologic conditions, a biomimetic ultrathin basement (biphasic elastic thin for air-liquid culture conditions, BETA) membrane (<1 µm) is developed with unique properties, including biocompatibility, permeability, and high elasticity (<10 kPa) for cell culturing under air-liquid interface and cyclic mechanical stretch conditions. The human-based triple coculture fibrosis model, which includes epithelial and endothelial cell lines combined with primary fibroblasts from idiopathic pulmonary fibrosis patients established on the BETA membrane, is integrated into a millifluidic bioreactor system (cyclic in vitro cell-stretch, CIVIC) with dose-controlled aerosolized drug delivery, mimicking inhalation therapy. The real-time measurement of cell/tissue stiffness (and compliance) is shown as a clinical biomarker of the progression/attenuation of fibrosis upon drug treatment, which is confirmed for inhaled Nintedanib-an antifibrosis drug. The mini-lung fibrosis model allows the combined longitudinal testing of pharmacodynamics and pharmacokinetics of drugs, which is expected to enhance the predictive capacity of preclinical models and hence facilitate the development of approved therapies for lung fibrosis.

Cell Adhesion Assessment Reveals a Higher Force per Contact Area on Fibrous Structures Compared to Flat Substrates.

The distribution and density of ligands have a determinant role in cell adhesion on planar substrates. At the same time, planar surfaces are nonphysiological for most cells, and cell behavior on planar and topographical surfaces is significantly different, with fibrous structures being the most natural environment for cells. Despite phenomenological examinations, the role of adhesion ligand density in the fibrous scaffold for cell adhesion strength has so far not been assessed. Here, we established a method to measure the amount of cell ligands on biofunctionalized electrospun meshes and planar substrate coatings with the same chemical composition. With this as a basis for systematic comparison and pure polyester as benchmark substrates, we have cultured L929 mouse fibroblasts and measured the adhesion force to surfaces of different chemistry and topography. In every case, having fibrous structures have led to an increased adhesion force per area also at a lower ligand density, which remarks the importance of such structures in a natural extracellular environment. Conversely, cells migrate more on planar surfaces than on the tested fibrous substrates. We thus established a platform to study cell-matrix interactions on different surfaces in a precise and reproducible manner as a new tool to assess and quantify cell-matrix interactions toward 3D scaffolds.

Antimicrobial and Wound-Healing Activities of Graphene-Reinforced Electrospun Chitosan/Gelatin Nanofibrous Nanocomposite Scaffolds.

This study aims at preparing electrospun chitosan/gelatin nanofiber scaffolds reinforced with different amounts of graphene nanosheets to be used as antibacterial and wound-healing scaffolds. Full characterization was carried out for the different fabricated scaffolds before being assessed for their antimicrobial activity against Escherichia coli and Staphylococcus aureus, cytotoxicity, and cell migration capacity. Raman and transmission electron microscopies confirmed the successful reinforcement of nanofibers with graphene nanosheets. Scanning electron microscopy and porosity revealed that nanofibers reinforced with 0.15% graphene nanosheets produced the least diameter (106 ± 30 nm) and the highest porosity (90%), in addition to their good biodegradability and swellability. However, the excessive increase in graphene nanosheet amount produced beaded nanofibers with decreased porosity, swellability, and biodegradability. Interestingly, nanofibers reinforced with 0.15% graphene nanosheets showed E. coli and S. aureus growth inhibition percents of 50 and 80%, respectively. The cell viability assay showed no cytotoxicity on human fibroblasts when cultured with either unreinforced or reinforced nanofibers. The cell migration was higher in the case of reinforced nanofibers when compared to the unreinforced nanofibers after 24 and 48 h, which is substantially associated with the great effect of the graphene nanosheets on the cell migration capability. Unreinforced and reinforced nanofibers showed cell migration results up to 93.69 and 97%, respectively, after 48 h.

Inducing Immunomodulatory Effects on Human Macrophages by Multifunctional NCO-sP(EO-stat-PO)/Gelatin Hydrogel Nanofibers.

Endowing materials and scaffolds with immunomodulatory properties has evolved into a very active field of research. However, combining such effects with multifunctionality regarding cell adhesion and manipulation is still challenging due to the intricate nature of cell-substrate interactions that require fine-tuning of scaffold properties. Here, we reported electrospinning of a well-known biopolymer, gelatin, together with six-arm star-shaped poly(ethylene oxide-stat-propylene oxide) prepolymer with isocyanate end groups (NCO-sP(EO-stat-PO)) as a reactive prepolymer cross-linker. Covalent coupling of two components during and after processing yielded a network of hydrogel fibers that was remarkably stable under aqueous and also proteolytic conditions without the need for extra cross-linking, with a significant increase in stability with increasing NCO-sP(EO-stat-PO) content. When seeded with human macrophages, cells adhered and spread on the fibers and were found highly viable after 7 days of culture across all scaffolds. Furthermore, hybrid fibrous meshes upregulated the expression of a prohealing gene, CD206, while downregulating proinflammatory genes, IL-1ß and IL-8. Markedly, NCO-sP(EO-stat-PO)-rich samples induced a significantly reduced release of proinflammatory cytokines, IL-1ß, IL-6, and IL-8. Finally, we successfully conjugated IL-4 to NCO-sP(EO-stat-PO) that effectively steered macrophages into a prohealing M2 type, demonstrating additional and robust control over the immunomodulatory feature of the scaffolds.

Appreciating the First Line of the Human Innate Immune Defense: A Strategy to Model and Alleviate the Neutrophil Elastase-Mediated Attack toward Bioactivated Biomaterials.

Biointerface engineering is a wide-spread strategy to improve the healing process and subsequent tissue integration of biomaterials. Especially the integration of specific peptides is one promising strategy to promote the regenerative capacity of implants and 3D scaffolds. In vivo, these tailored interfaces are, however, first confronted with the innate immune response. Neutrophils are cells with pronounced proteolytic potential and the first recruited immune cells at the implant site; nonetheless, they have so far been underappreciated in the design of biomaterial interfaces. Herein, an in vitro approach is introduced to model and analyze the neutrophil interaction with bioactivated materials at the example of nano-bioinspired electrospun surfaces that reveals the vulnerability of a given biointerface design to the contact with neutrophils. A sacrificial, transient hydrogel coating that demonstrates optimal protection for peptide-modified surfaces and thus alleviates the immediate cleavage by neutrophil elastase is further introduced.

Bioactive Electrospun Fibers: Fabrication Strategies and a Critical Review of Surface-Sensitive Characterization and Quantification.

Fabricating a porous scaffold with high surface area has been a major strategy in the tissue engineering field. Among the many fabrication methods, electrospinning has become one of the cornerstone techniques due to its enabling the fabrication of highly porous fibrous scaffolds that are of natural or synthetic origin. Apart from the basic requirements of mechanical stability and biocompatibility, scaffolds are further expected to embody functional cues that drive cellular functions such as adhesion, spreading, proliferation, migration, and differentiation. There are abundant distinct approaches to introducing bioactive molecules to have a control over cellular functions. However, the lack of a thorough understanding of cell behavior with respect to the availability and spatial distribution of the bioactive molecules in 3D fibrous scaffolds is yet to be addressed. The rational selection of proper sets of characterization techniques would essentially impact the interpretation of the cell-scaffold interactions. In this timely Review, we summarize the most popular methods to introduce functional compounds to electrospun fibers. Thereafter, the strength and limitations of the conventional characterization methods are highlighted. Finally, the potential and applicability of emerging characterization techniques such as high-resolution/correlative microscopy approaches are further discussed.

A Bioinspired in vitro Lung Model to Study Particokinetics of Nano-/Microparticles Under Cyclic Stretch and Air-Liquid Interface Conditions.

Evolution has endowed the lung with exceptional design providing a large surface area for gas exchange area (ca. 100 m2) in a relatively small tissue volume (ca. 6 L). This is possible due to a complex tissue architecture that has resulted in one of the most challenging organs to be recreated in the lab. The need for realistic and robust in vitro lung models becomes even more evident as causal therapies, especially for chronic respiratory diseases, are lacking. Here, we describe the Cyclic I n VI tro Cell-stretch (CIVIC) "breathing" lung bioreactor for pulmonary epithelial cells at the air-liquid interface (ALI) experiencing cyclic stretch while monitoring stretch-related parameters (amplitude, frequency, and membrane elastic modulus) under real-time conditions. The previously described biomimetic copolymeric BETA membrane (5 µm thick, bioactive, porous, and elastic) was attempted to be improved for even more biomimetic permeability, elasticity (elastic modulus and stretchability), and bioactivity by changing its chemical composition. This biphasic membrane supports both the initial formation of a tight monolayer of pulmonary epithelial cells (A549 and 16HBE14o-) under submerged conditions and the subsequent cell-stretch experiments at the ALI without preconditioning of the membrane. The newly manufactured versions of the BETA membrane did not improve the characteristics of the previously determined optimum BETA membrane (9.35% PCL and 6.34% gelatin [w/v solvent]). Hence, the optimum BETA membrane was used to investigate quantitatively the role of physiologic cyclic mechanical stretch (10% linear stretch; 0.33 Hz: light exercise conditions) on size-dependent cellular uptake and transepithelial transport of nanoparticles (100 nm) and microparticles (1,000 nm) for alveolar epithelial cells (A549) under ALI conditions. Our results show that physiologic stretch enhances cellular uptake of 100 nm nanoparticles across the epithelial cell barrier, but the barrier becomes permeable for both nano- and micron-sized particles (100 and 1,000 nm). This suggests that currently used static in vitro assays may underestimate cellular uptake and transbarrier transport of nanoparticles in the lung.

Electrospun nanofiber mesh with fibroblast growth factor and stem cells for pelvic floor repair.

Surgical outcome following pelvic organ prolapse (POP) repair needs improvement. We suggest a new approach based on a tissue-engineering strategy. In vivo, the regenerative potential of an electrospun biodegradable polycaprolactone (PCL) mesh was studied. Six different biodegradable PCL meshes were evaluated in a full-thickness abdominal wall defect model in 84 rats. The rats were assigned into three groups: (1) hollow fiber PCL meshes delivering two dosages of basic fibroblast growth factor (bFGF), (2) solid fiber PCL meshes with and without bFGF, and (3) solid fiber PCL meshes delivering connective tissue growth factor (CTGF) and rat mesenchymal stem cells (rMSC). After 8 and 24 weeks, we performed a histological evaluation, quantitative analysis of protein content, and the gene expression of collagen-I and collagen-III, and an assessment of the biomechanical properties of the explanted meshes. Multiple complications were observed except from the solid PCL-CTGF mesh delivering rMSC. Hollow PCL meshes were completely degraded after 24 weeks resulting in herniation of the mesh area, whereas the solid fiber meshes were intact and provided biomechanical reinforcement to the weakened abdominal wall. The solid PCL-CTGF mesh delivering rMSC demonstrated improved biomechanical properties after 8 and 24 weeks compared to muscle fascia. These meshes enhanced biomechanical and biochemical properties, demonstrating a great potential of combining tissue engineering with stem cells as a new therapeutic strategy for POP repair. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:48-55, 2020.

Bioadhesive anisotropic nanogrooved microfibers directing three-dimensional neurite extension.

Neurodegenerative diseases and acute nerve injuries are becoming global clinical problems. Engineering three-dimensional (3D), anisotropic neural cellular structures in vitro is therefore desirable in the regenerative medicine research community. Here, we present, for the first time, a single-step, facile but delicate, fabrication of a 3D macroporous microfibrous scaffold with both anisotropic nanogrooved topography and in situ functionalization with a mussel inspired bioadhesive, poly(norepinephrine) (pNE). Specifically, immiscible blends of polycaprolactone (PCL) and polyethylene oxide (PEO) were electrospun into a grounded coagulation bath containing the precursor of pNE. Upon jet entrance in the bath, both phase-separation-driven longitudinal nanotopography and in situ pNE surface functionalization were introduced on individual microfibers that were packed into a 3D macroporous structure. The resulting scaffold significantly promoted 3D neurite extension capacity, 8-fold higher neurite extension over the isotropic counterpart, demonstrating that such a scaffold has great promise in 3D neural cell culture for nerve tissue modelling and engineering.

Electrospun biodegradable microfibers induce new collagen formation in a rat abdominal wall defect model: A possible treatment for pelvic floor repair?

Half of the female population over age 50 years will experience pelvic organ prolapse. We suggest a new approach based on tissue engineering principles to functionally reconstruct the anatomical structures of the pelvic floor. The aim of this study is to investigate the mechanical performance and effect on collagen and elastin production of a degradable mesh releasing basic fibroblast growth factor (bFGF). Implantation of biodegradable mesh with or without bFGF in their core has been conducted in 40 rats in an abdominal wall defect model. Samples were explanted after 4, 8, and 24 weeks, and tested for mechanical properties and the composition of connective tissue. The study showed an increase in mRNA expression for collagen-I (p = 0.0060) and collagen-III (p = 0.0086) in the 4 weeks group with bFGF. The difference was equalized at 8 and 24 weeks. No difference was found at any time for protein amount for collagen-I, collagen-III, and fibronectin. The amount of collagen decreased from 4 to 24 weeks but the fraction of collagen increased. The maximal load of the newly formed tissue showed no effect of bFGF at any time. Exclusively, histology showed a limited ingrowth of collagen fibers after 4 weeks with bFGF but signs of elastin fibers were seen at 24 weeks. The investigation showed that a biodegradable mesh promotes tissue formation with a promising strength. The mesh with bFGF did not represent any advantage on either long or short term in comparison to the mesh without bFGF. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 680-688, 2018.

Mussel Inspired Polynorepinephrine Functionalized Electrospun Polycaprolactone Microfibers for Muscle Regeneration.

Electrospun scaffolds with excellent mechanical properties, high specific surface area and a commendable porous network are widely used in tissue engineering. Improving the hydrophilicity and cell adhesion of hydrophobic substrates is the key point to enhance the effectiveness of electrospun scaffolds. In this study, polycaprolactone (PCL) fibrous membranes with appropriate diameter were selected and coated by mussel-inspired poly norepinephrine (pNE). And norepinephrine is a catecholamine functioning as a hormone and neurotransmitter in the human brain. The membrane with smaller diameter fibers, a relative larger specific surface area and the suitable pNE functionalization provided more suitable microenvironment for cell adhesion and proliferation both in vitro and in vivo. The regenerated muscle layer can be integrated well with fibrous membranes and surrounding tissues at the impaired site and thus the mechanical strength reached the value of native tissue. The underlying molecular mechanism is mediated via inhibiting myostatin expression by PI3K/AKT/mTOR hypertrophy pathway. The properly functionalized fibrous membranes hold the potential for repairing muscle injuries. Our current work also provides an insight for rational design and development of better tissue engineering materials for skeletal muscle regeneration.

Nanotopography featured polycaprolactone/polyethyleneoxide microfibers modulate endothelial cell response.

Among many physical properties, surface nanotopography has been found to strongly affect cell adhesion, migration and other functions. Accurate biological interpretation requires the nanotopography to be presented in a three-dimensional (3D) micro-environment. Herein, immiscible blends of polycaprolactone (PCL)/polyethyleneoxide (PEO) were electrospun into a grounded coagulation bath, resulting in macroporous microfibers with nanotopography featured surfaces. Variations in PCL/PEO ratios enabled tunable surface nanotopographic structures, from longitudinal submicron grooves to transverse nano-lamellae. Chemical composition, crystallinity and quantitative nanomechanical analysis confirmed that the interplay of the two semi-crystalline immiscible polymers and the pairing of miscible solvents/non-solvents in both the electrospinning solution and the bath solution were critical for the formation of the secondary structure. It was found that the nanotopography features promoted the proliferation of human umbilical vein endothelial cells (HUVECs) compared with their smooth film counterparts. An analysis of the cell adhesion related markers, vinculin and phosphorylated focal adhesion kinase (pFAK), further revealed that the nanotopographies enhanced the nascent adhesion complex formation compared with smooth PCL fibers, even in the scaffolds with a high PEO content, which is often considered as a non-adhesive material.

Three-Dimensional Polydopamine Functionalized Coiled Microfibrous Scaffolds Enhance Human Mesenchymal Stem Cells Colonization and Mild Myofibroblastic Differentiation.

Electrospinning has been widely applied for tissue engineering due to its versatility of fabricating extracellular matrix (ECM) mimicking fibrillar scaffolds. Yet there are still challenges such as that these two-dimensional (2D) tightly packed, hydrophobic fibers often hinder cell infiltration and cell-scaffold integration. In this study, polycaprolactone (PCL) was electrospun into a grounded coagulation bath collector, resulting in 3D coiled microfibers with in situ surface functionalization with hydrophilic, catecholic polydopamine (pDA). The 3D scaffolds showed biocompatibility and were well-integrated with human bone marrow derived human mesenchymal stem cells (hMSCs), with significantly higher cell penetration depth compared to that of the 2D PCL microfibers from traditional electrospinning. Further differentiation of human mesenchymal stem cells (hMSCs) into fibroblast phenotype in vitro indicates that, compared to the stiff, tightly packed, 2D scaffolds which aggravated myofibroblasts related activities, such as upregulated gene and protein expression of α-smooth muscle actin (α-SMA), 3D scaffolds induced milder myofibroblastic differentiation. The flexible 3D fibers further allowed contraction with the well-integrated, mechanically active myofibroblasts, monitored under live-cell imaging, whereas the stiff 2D scaffolds restricted that.

Poly(norepinephrine) as a functional bio-interface for neuronal differentiation on electrospun fibers.

Based on the catecholic chemistry of a mussel inspired coating, norepinephrine (NE), a catecholamine found both in neurotransmitters and mussel adhesive proteins, was for the first time applied as a unique bio-interface integrating multi-functions facilitating PC12 neuron-like differentiation. A uniform, ultra-smooth pNE coating was achieved on electrospun submicron PLCL fibers, proven by surface characterization. The introduced catechol groups from pNE were further used to not only anchor collagen to enhance cell adhesion but also localize nerve growth factor to promote neuron-like differentiation. The obtained pNE-collagen coating was found to be a superior substrate for PC12 differentiation, confirmed by both cellular toxicity/viability assays and immunochemical staining. The aligned PLCL fiber conformation further steered neurite formation along the fiber direction and contributed to neurite extension and increased the number of neurites per cell body. This facile pNE coating might lead to a more efficient use of growth factor, drugs and other bioactive molecules with lower loading dosage and sustained activity resulting in enhanced therapeutic effects and decreased adverse effects.

hiPS-MSCs differentiation towards fibroblasts on a 3D ECM mimicking scaffold.

Fibroblasts are ubiquitous cells that constitute the stroma of virtually all tissues and play vital roles in homeostasis. The poor innate healing capacity of fibroblastic tissues is attributed to the scarcity of fibroblasts as collagen-producing cells. In this study, we have developed a functional ECM mimicking scaffold that is capable to supply spatial allocation of stem cells as well as anchorage and storage of growth factors (GFs) to direct stem cells differentiate towards fibroblasts. Electrospun PCL fibers were embedded in a PEG-fibrinogen (PF) hydrogel, which was infiltrated with connective tissue growth factor (CTGF) to form the 3D nanocomposite PFP-C. The human induced pluripotent stem cells derived mesenchymal stem cells (hiPS-MSCs) with an advance in growth over adult MSCs were applied to validate the fibrogenic capacity of the 3D nanocomposite scaffold. The PFP-C scaffold was found not only biocompatible with the hiPS-MSCs, but also presented intriguingly strong fibroblastic commitments, to an extent comparable to the positive control, tissue culture plastic surfaces (TCP) timely refreshed with 100% CTGF. The novel scaffold presented not only biomimetic ECM nanostructures for homing stem cells, but also sufficient cell-approachable bio-signaling cues, which may synergistically facilitate the control of stem cell fates for regenerative therapies.

Electrospun PCL/PEO coaxial fibers for basic fibroblast growth factor delivery.

The poor innate healing capacity of fibroblastic tissues, such as pelvic floor fascia, is attributed to the scarcity of fibroblasts to produce collagen, as the main collagen producing cells. Coaxial electrospun PCL/PEO fibers containing basic fibroblast growth factor (FGF-2) were evaluated for the local and temporal delivery of FGF-2 for promoting fibroblast proliferation. PCL/PEO coaxial fibers with a highly porous surface were successfully developed using coaxial electrospinning. The diameter of the PCL/PEO microfibers produced by coaxial electrospinning could be tuned by the electrospinning parameters. XPS surface chemistry probing and CA wettability analysis confirmed that the outer surface of the coaxial fibers is PCL. The protein was successfully encapsulated and a sustained release was observed over more than 9 days. In vitro, PCL/PEO coaxial fibers supported fibroblast cell adhesion. In addition, PCL/PEO coaxial fibers containing FGF-2 significantly enhanced fibroblast cell viability and proliferation. Further, Coll-I expression was significantly expressed after day 1 while down-regulated after day 9 compared to the control group. These results indicate that coaxial polymeric fibers allow local and sustained growth factor delivery with prolonged efficacy and longevity for connective tissue regeneration.