The study addressed an apparent contradiction in the biology of p75NTR, a member of the tumor necrosis factor receptor superfamily that regulates cell death, survival and differentiation in a context-dependent manner. Earlier experiments had associated the receptor with enhanced osteogenesis in some mesenchymal cell systems, while other studies reported inhibitory effects. The researchers examined p75NTR-knockout mice and ectomesenchymal stem cells, or EMSCs, isolated from the maxillofacial tissue of E16.5 embryos.

Knockout animals were smaller than their wild-type littermates and showed delayed skeletal development, with more cartilage and less mineralized bone. Micro-computed tomography also revealed reduced bone volume fraction, bone mineral density and trabecular number in the femur and mandible. The knockout phenotype was not confined to a single differentiation assay.

Flow cytometry showed that wild-type and knockout EMSCs expressed the mesenchymal stem-cell markers CD29, CD90 and Sca-1 at high frequencies while showing little expression of the hematopoietic marker CD45, supporting their characterization as mesenchymal stem-cell-like populations. However, knockout EMSCs displayed weaker multilineage differentiation than wild-type cells. After osteogenic induction, knockout cultures formed fewer calcium deposits, while adipogenic and chondrogenic assays showed fewer lipid droplets and cartilage nodules.

These observations placed the therapeutic work in the context of a broad developmental impairment associated with the loss of p75NTR. The researchers isolated extracellular vesicles by differential ultracentrifugation from cultures of wild-type and knockout EMSCs. Wild-type vesicles had a mean particle diameter of 142.6 ± 64.9 nanometres, and knockout-derived vesicles measured 150.5 ± 95.8 nanometres.

Transmission electron microscopy showed rounded or cup-shaped structures in both preparations. Both vesicle types contained the exosomal markers TSG101 and CD9 but lacked detectable calnexin, an endoplasmic-reticulum marker used as a negative control. Fluorescently labelled wild-type vesicles entered recipient EMSCs and accumulated in the perinuclear region at 12 and 24 hours.

In cell-culture assays, wild-type vesicles increased knockout-cell viability in a concentration-dependent manner; 50 micrograms per millilitre produced a 24% increase in viability over the control group on day five. At that dose, proliferation of treated knockout cells approached the wild-type level by day five, although the authors described the effect as a partial restoration of proliferative capacity. Alkaline phosphatase staining indicated stronger early differentiation, while Alizarin Red staining after 21 days showed more calcium-nodule formation.

At 50 micrograms per millilitre, the treated knockout cells expressed several osteogenic markers at levels similar to those of untreated wild-type cells. Conversely, vesicles derived from knockout EMSCs suppressed collagen I, alkaline phosphatase and Runx2 expression in wild-type cells. The reciprocal pattern shows that vesicle activity differed according to the donor cells’ p75NTR state, but the experiments did not identify which exosomal cargo was responsible.

To improve local retention and duration of treatment, the team encapsulated wild-type vesicles in the aqueous core of coaxial electrospun fibres. The shell combined silk fibroin and polycaprolactone, while the core contained polyvinyl alcohol and the vesicles. After screening several shell compositions and loading concentrations, the authors selected an SF/PCL ratio of 2:5 and a vesicle concentration of 10 milligrams per millilitre.

Scanning electron microscopy showed relatively uniform fibres in the selected formulation, with a mean diameter of 146.8 ± 27.6 nanometres before vesicle loading and 156.9 ± 40.5 nanometres afterward. Dual fluorescence confirmed that the vesicle-containing solution occupied the core of the fibres. Release followed a three-stage pattern, with an initial burst followed by more gradual delivery over 28 days.

By day 28, cumulative release reached 56.88 ± 2.01% for the selected formulation, and released vesicles were taken up by knockout EMSCs during culture. The membrane was biocompatible with knockout EMSCs: live/dead staining detected no dead cells after three days, and the exosome-loaded membrane accelerated proliferation from day three through day five. At seven and 14 days, alkaline-phosphatase activity was higher with the loaded membrane than with the unloaded membrane or control, while the loaded membrane group showed the highest observed activity.

At 21 days, the loaded membrane produced the greatest mineral deposition, and immunofluorescence showed stronger collagen I and osteopontin signals. RNA sequencing of knockout cells cultured under osteogenic conditions identified 186 upregulated and 90 downregulated genes in the loaded-membrane group compared with the unloaded membrane, using thresholds of absolute log2 fold change of at least 1 and adjusted p below 0.05. Pathway analysis highlighted PI3K/Akt signaling, while enriched biological processes included skeletal development, ossification and osteoblast differentiation.

Pharmacological experiments implicated both PI3K/Akt and ERK1/2 in the response. The loaded membrane increased phosphorylation of Akt and ERK1/2 and raised the measured spatial association between the two phosphorylated proteins. The PI3K activator 740Y-P further increased Akt phosphorylation and osteogenic outputs, whereas the PI3K inhibitor LY294002 reduced Akt phosphorylation, alkaline-phosphatase activity, mineralization and expression of osteopontin and osteocalcin.

The ERK inhibitor PD98059 reduced ERK1/2 phosphorylation without significantly changing Akt phosphorylation. Modulating PI3K/Akt produced corresponding changes in ERK1/2 phosphorylation. These results support a directional relationship in which PI3K/Akt influences ERK1/2 activation, but no genetic experiment was performed to establish the molecular link or distinguish direct from indirect effects.

In vivo, 8-week-old knockout mice received 3-millimetre cranial defects and were assigned to loaded-membrane, unloaded-membrane, free-vesicle or no-treatment groups, with wild-type mice serving as controls. Six male mice were included in each group at the four- and eight-week observation points, for 60 mice in the full study. Each loaded membrane contained 100 micrograms of vesicle protein; the free-vesicle group received repeated local injections, with the cumulative dose reaching 100 micrograms by the eighth injection.

Micro-computed tomography at four and eight weeks showed greater mineralized bone in defects treated with the loaded membrane than in the other knockout groups. At eight weeks, its bone surface area was 31.91 square millimetres, compared with 20.45 for free vesicles and 20.03 for the unloaded membrane. Maximum load reached 4.188 ± 0.316 newtons, significantly above the free-vesicle and unloaded-membrane groups and not significantly different from the wild-type value of 4.057 ± 0.357 newtons.

Untreated knockout defects lacked sufficient mineralized tissue for reliable load testing. Histology was consistent with the imaging findings. By eight weeks, defects supported by the loaded membrane showed continuous bridging, thicker and more compact trabeculae, reduced inter-trabecular spaces and areas resembling lamellar bone, whereas the other treated groups retained incomplete bridging and fibrous tissue.

Collagen I and osteopontin staining was strongest after membrane-mediated delivery. Immunohistochemistry indicated increased phosphorylated Akt and ERK1/2 in the loaded-membrane group, together with higher ATF4 and Runx2 expression. Examination of the heart, liver, spleen, lungs and kidneys found no reported inflammation or pathological damage at eight weeks.

The experiments therefore support a preclinical cell-free strategy combining exosome-mediated signaling with local biomaterial delivery, but they do not establish efficacy in people. The small-animal model, male-only cohort, eight-week observation period, absence of long-term and large-animal safety data, and inability of the protein-based release assay to distinguish intact vesicles from degraded exosomal protein remain important limitations.

Subject of Research: Preclinical testing of exosome-loaded nanofibrous membranes for p75NTR-associated bone-regeneration deficiency

Article Title: EMSCs-derived exosomes rescue p75NTR −/− -mediated osteogenesis deficiency via PI3K/Akt/ERK1/2 signaling pathways

Article References: Ye, J., Jin, X., Huang, Y., Khup, P. L., Yu, Y., Cheng, M., Yuan, Q., Xu, D., Nie, X., & Cheng, G. (2026). EMSCs-derived exosomes rescue p75NTR−/− -mediated osteogenesis deficiency via PI3K/Akt/ERK1/2 signaling pathways. Materials Today Bio, 41, Article 103755. https://doi.org/10.1016/j.mtbio.2026.103755

Image Credits: AI Generated

DOI: 10.1016/j.mtbio.2026.103755

Keywords: p75NTR, extracellular vesicles, bone regeneration, osteogenesis, nanofibrous membranes, PI3K/Akt signaling, ERK1/2 signaling, cranial defects, ectomesenchymal stem cells, EMSCs-derived, exosomes, rescue

The metals in question are among the most frequently detected pollutants in industrial wastewater. Lead and copper ions originate from mining, electroplating, battery manufacturing, and metallurgical processing, and even at trace concentrations they can trigger neurological, renal, and cardiovascular disorders in humans while accumulating in aquatic food chains. Conventional remediation techniques such as chemical precipitation, ion exchange, coagulation–flocculation, and activated carbon adsorption all carry drawbacks, including high operational costs, the generation of contaminated sludge, and poor performance at low metal concentrations. Membrane-based separation has emerged as an appealing alternative because of its operational simplicity, compact footprint, and potential for continuous treatment, but the performance of any membrane depends heavily on how it is made.

That is where the Brazilian team’s choice of fabrication method becomes significant. Most nanofiber membranes described in the literature are produced by electrospinning, a technique that draws fibers from a polymer solution using high voltage. Electrospinning produces excellent fibers but suffers from low production rates and high energy consumption, which have limited its industrial adoption. The researchers instead used solution blow spinning, or SBS, a technique that replaces the electric field with a stream of compressed air. In SBS, the polymer solution is fed through a concentric nozzle while pressurized air simultaneously stretches the emerging jet into fine fibers, which are collected on a rotating drum. The method requires no high-voltage equipment, offers higher throughput, and still yields highly porous micro- and nanofibrous mats, making it an attractive route to membranes that could one day be manufactured at scale.

To give the inherently hydrophobic PVDF an affinity for metal ions, the team dispersed montmorillonite clay into the polymer solution at loadings ranging from 3 to 30 percent by weight relative to the polymer. Montmorillonite is a layered silicate with a high specific surface area and a substantial cation exchange capacity. Its negatively charged interlayer spaces and surface hydroxyl groups attract divalent metal cations such as Pb²⁺ and Cu²⁺ through electrostatic interactions and ion exchange, effectively turning the clay particles scattered through the fibrous network into a dense population of active adsorption sites. The researchers fabricated two families of membranes: pristine PVDF nanofiber mats and PVDF–clay composites, keeping the solution volume constant at five milliliters for every production run so that thickness differences could be attributed to the clay content itself.

Scanning electron microscopy confirmed that the process worked as intended. The membranes displayed uniform, smooth, cylindrical fibers with only occasional bead-like imperfections, a sign that the spinning parameters—30 percent polymer concentration, a solution flow rate of 76 microliters per hour, air pressure of 140 kilopascals, a working distance of 21 centimeters, and a collector speed of 80 revolutions per minute—were well chosen. X-ray diffraction revealed the characteristic crystalline phases of PVDF alongside clay-derived peaks corresponding to quartz and aluminum oxide, confirming that the filler was genuinely incorporated into the fibers. At the highest loading of 30 percent, however, clay agglomerates began to appear, producing structural defects, thinner and more fragile films, and, above that threshold, outright clogging and failure of fiber formation.

Before testing adsorption, the team determined the point of zero charge of the membranes, the pH at which the surface carries equal numbers of positive and negative charges. This value came out at 6.9 in pure water and 6.4 in a water–ethanol mixture, and the adsorption experiments were conducted near these values to minimize interference from the liquid medium. Batch tests, in which membrane pieces of roughly 50 milligrams were stirred with 20 milliliters of metal solution at 5 milligrams per liter, showed a familiar pattern: rapid uptake in the first hours as abundant adsorption sites were available, followed by a gradual slowdown as those sites filled. Adsorption was consistently higher in the water–ethanol mixture, which the authors attribute to ethanol acting like a surfactant, lowering the surface tension of water and helping the solution wet the hydrophobic polymer and penetrate the spaces between fibers to reach buried clay sites.

The kinetic and equilibrium analysis told a coherent mechanistic story. A pseudo-first-order model fit the data poorly, with correlation coefficients as low as 0.674, whereas the pseudo-second-order model tracked the experiments closely in both media, indicating that the rate-limiting step involves chemical interaction—chemisorption—between the metal ions and functional sites on the clay surface. Equilibrium data were best described by the Langmuir isotherm, which assumes monolayer adsorption on a finite number of identical sites, and the calculated maximum adsorption capacities matched the experimental values well. The dimensionless separation factor derived from the Langmuir constant was below one in every test, confirming that adsorption of both metals is thermodynamically favorable. Still, the Langmuir constants were low, between 0.044 and 0.298, and more than half of the dissolved metal remained in solution at equilibrium in the static tests—a limitation the researchers trace directly to the hydrophobic PVDF matrix, which impedes ion access to clay sites in stagnant water.

The picture changed dramatically when the membranes were operated as filters. In dead-end filtration experiments, in which the metal solution was forced through a 12.5 square centimeter membrane area under roughly one bar of pressure from a vacuum pump, removal efficiencies soared above 90 percent, with the membrane containing 10 percent clay achieving 92 percent copper removal in aqueous medium. The lead removal performance matched that of comparable electrospun membranes reported in the literature, but with the advantage of a faster, cheaper production process. Even pure PVDF removed a meaningful fraction of the metals, demonstrating that the porous fibrous architecture itself contributes to capture. The combined mechanism—physical retention by the dense fiber network working in tandem with adsorption at clay sites under pressure-driven flow—proved far more effective than batch adsorption alone, because the applied pressure overcomes the wetting resistance that limits ion penetration in static conditions.

Intriguingly, more clay was not always better. At 30 percent loading, filtration performance deteriorated: aggregates identified by energy-dispersive X-ray analysis acted as bypass routes that let water, and the metals it carried, slip through without contacting adsorbent sites, while the weakened mechanical properties of the highly loaded membranes allowed pores to widen under pressure. The study also revealed a classic trade-off between selectivity and permeability, with thicker, less permeable membranes delivering the highest selectivity. The authors are candid about the work’s limits: no regeneration or reuse studies were performed, long-term stability remains untested, and performance in multicomponent, real-world wastewater has yet to be assessed. Future work, they note, should address membrane reuse, metal leaching, permeability–selectivity trade-offs, and continuous-flow operation. Even so, the message is clear: solution blow spinning can rapidly produce PVDF–clay nanofiber membranes that excel as filtration materials, and with moderate clay loadings of around 10 percent, they offer a promising, scalable route to stripping toxic lead and copper from contaminated water.

Subject of Research: PVDF–montmorillonite clay nanofibrous membranes produced by solution blow spinning for the removal of lead and copper ions from contaminated water

Article Title: Removal of Pb and Cu metals by PVDF/clay membranes obtained through solution blow spinning technique

Article References: da Cruz Dias, G., Zadorosny, L., Sanches, A. O., dos Santos, M. C., & Malmonge, L. F. (2026). Removal of Pb and Cu metals by PVDF/clay membranes obtained through solution blow spinning technique. Journal of Materials Science: Polymers, 1(1), Article 18. https://doi.org/10.1007/s44493-026-00020-7

Image Credits: AI Generated

DOI: 10.1007/s44493-026-00020-7

Keywords: solution blow spinning, PVDF, montmorillonite clay, nanofibrous membranes, heavy metal removal, lead, copper, water treatment, adsorption kinetics, Langmuir isotherm, membrane filtration, wastewater remediation