Black Phosphorus Nanoparticles Boost Osteogenic Differentiation of Ectodermal Mesenchymal Stem Cells via TG2 Upregulation

Abstract

Black phosphorus nanoparticles (BPs) at bio‑safe concentrations activate transglutaminase 2 (TG2) and upregulate extracellular matrix (ECM) components, thereby accelerating osteogenic differentiation of ectodermal mesenchymal stem cells (EMSCs). BPs exhibit excellent biocompatibility and biodegradability, making them promising additives for bone‑tissue engineering scaffolds. In vitro, BPs (2–4 µg mL⁻¹) significantly enhanced osteogenic markers (RUNX2, ALP, COL‑I, OCN, OPN, BMP‑2) without affecting proliferation. TG2 inhibition abrogated these effects, confirming a TG2‑dependent mechanism. These findings support the clinical potential of BPs in bone regeneration strategies.

Introduction

Effective bone repair demands materials that are osteoconductive, osteoinductive, and osteointegrative. Traditional autografts and allografts have limited availability and carry infection risks. Recent advances in biomaterials—particularly ion‑releasing ceramics and bioactive polymers—have improved outcomes, yet a material that naturally supplies phosphate and promotes mineralization remains elusive.

Black phosphorus (BP), a layered two‑dimensional allotrope, has attracted attention for its tunable bandgap, high carrier mobility, and rapid biodegradation to benign phosphate ions. Prior work has shown BP nanosheets enhance mineralization by providing phosphate donors, but the cellular mechanisms remain poorly understood. Ectodermal mesenchymal stem cells (EMSCs), harvested from adult nasal mucosa, offer a readily accessible, non‑invasive source of multipotent cells capable of osteogenic differentiation.

We investigated whether BPs could act as osteo‑inductive cues for EMSCs and delineated the underlying pathways, focusing on TG2—a key enzyme that crosslinks ECM proteins and regulates osteoblast differentiation.

Materials and Methods

Black Phosphorus Nanoparticle Preparation

Bulk BP (20 mg) was dispersed in saturated NaOH/NMP, mechanically ground, and ultrasonicated (6 h) in an ice bath. After filtration (100 µm) and high‑speed centrifugation (13 000 rpm, 10 min, 4 °C), a stable BP suspension was obtained. Particle size (132 nm) and zeta potential (−23.7 mV) were confirmed by DLS; SEM confirmed a 100–150 nm sheet morphology. Raman and XRD spectra verified BP phase purity.

EMSC Isolation and Culture

Adult Sprague‑Dawley rats were anesthetized, and nasal septal mucosa was harvested. Tissue was digested (0.25 % trypsin, 37 °C, 25 min) and passed through a 100 µm sieve. Cells were expanded in DMEM/F12 + 10 % FBS, 100 U mL⁻¹ penicillin, 100 µg mL⁻¹ streptomycin. Passage 3 cells were used for all assays; immunofluorescence confirmed >95 % nestin and vimentin expression.

Osteogenic Differentiation

EMSCs were seeded at 3 000 cells cm⁻² and induced with osteogenic medium (10 % FBS, 0.1 mM dexamethasone, 0.2 mM ascorbic acid) for 14 days (no β‑glycerophosphate to avoid confounding). Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining assessed early and late differentiation, respectively. RT‑qPCR measured RUNX2, ALP, COL‑I, OCN, OPN, BMP‑2 at days 7 and 14.

Cell Viability and Proliferation

CCK‑8 assays evaluated BPs toxicity (0–512 µg mL⁻¹) after 24 h. Ki‑67 immunostaining quantified proliferation. Concentrations ≤32 µg mL⁻¹ were non‑cytotoxic; 2 and 4 µg mL⁻¹ were chosen for functional studies.

Mechanistic Studies

Western blotting quantified TG2, fibronectin (FN), laminin (LN), OCN, OPN, COL‑I. TG2 neutralizing antibody (10 µg mL⁻¹) was applied to confirm pathway involvement. Statistical analysis used Student’s t‑test and ANOVA (p < 0.05). All experiments were performed in triplicate.

Results

Characterization of BPs

DLS showed a narrow size distribution (100–200 nm, peak 132 nm). Zeta potential of −23.7 mV confirmed colloidal stability. SEM images matched DLS data. Raman peaks at 362.3, 438.5, 466.9 cm⁻¹ confirmed BP structure; XRD indicated an average crystallite size of 102.7 nm.

EMSC Phenotype

Immunofluorescence of nasal mucosa revealed nestin⁺ cells in the lamina propria. Passage 3 EMSCs displayed fibroblastic morphology and >95 % nestin/vimentin positivity. Under osteogenic induction, ARS staining showed mineral deposits at day 28; adipogenic induction yielded Oil Red O staining at day 21.

Biocompatibility

CCK‑8 assays indicated no significant viability loss up to 32 µg mL⁻¹. Ki‑67 staining confirmed unchanged proliferation at 2 and 4 µg mL⁻¹.

Osteogenic Enhancement by BPs

ARS staining revealed larger, more regular calcium nodules in 2 and 4 µg mL⁻¹ groups compared to control (p < 0.05). ALP activity increased at day 7 in BP‑treated cells (p > 0.05 between 2 vs 4 µg mL⁻¹). RT‑qPCR showed significant upregulation of RUNX2, ALP, COL‑I, OCN, OPN, BMP‑2 at day 14 in BP groups (p < 0.01).

TG2‑Dependent Mechanism

Western blotting demonstrated a ~2‑fold increase in intracellular and extracellular TG2 in BP‑treated cells. FN and LN levels were also elevated. TG2 neutralization abolished BP‑induced ARS deposition, ALP activity, and expression of OCN, OPN, COL‑I. These data confirm that BPs promote osteogenesis through TG2 upregulation and ECM remodeling.

Discussion

Our study demonstrates that BPs are safely incorporated into the osteogenic milieu of EMSCs at concentrations below 32 µg mL⁻¹. The nanoparticles provide a rapid source of phosphate, facilitating mineralization, while simultaneously activating TG2—a multifunctional enzyme that crosslinks ECM proteins and modulates integrin signaling. The resulting ECM enrichment (FN, LN, COL‑I) likely enhances cell–matrix interactions, further driving osteogenic commitment. These findings align with previous reports that BP nanomaterials accelerate bone repair by delivering phosphate ions and stimulating osteoblast activity.

While BP’s susceptibility to oxidation can limit stability, its rapid biodegradation to harmless phosphate reduces long‑term accumulation risks. Nonetheless, in vivo studies are required to confirm safety and efficacy in animal models and to optimize scaffold designs for clinical translation.

Conclusion

Black phosphorus nanoparticles, produced via a scalable grinding‑ultrasonication process, safely enhance TG2‑mediated osteogenic differentiation of EMSCs in vitro. These results support their incorporation into tissue‑engineered bone scaffolds. Future preclinical work should address pharmacokinetics, biodistribution, and therapeutic outcomes in bone defect models.

Availability of Data and Materials

Datasets are available from the corresponding author upon reasonable request.