Treating Bone Cancer and Repairing the Damage: Why This Study Matters

Bone tumors, especially osteosarcoma, often affect children and adolescents and have a relatively poor long-term survival rate. The standard approach in the clinic is:

• Surgical removal of the tumor

• Chemotherapy before and/or after surgery

However, this creates two big problems:

1. Residual cancer cells
   Even after careful surgery, some tumor cells can remain around the resection site and cause local recurrence.

2. Large bone defects
   When surgeons remove a bone tumor, they usually have to remove a significant amount of healthy bone around it for safety margins. This creates a large defect in the bone that must be repaired for the patient to regain normal function.

The current “gold standard” to fill these defects is autologous bone grafting, which means taking bone from another site in the same patient (for example, the pelvis) and implanting it in the defect. But this approach has serious limitations:

• The amount of donor bone is limited.

• Harvesting bone can cause pain, infection, and other complications.

• It is not easy to treat both the tumor and the defect with the same material.

Because of these issues, researchers have been working in two directions:

• Bone tissue engineering – using scaffolds and biomaterials to support bone repair and regeneration.

• Precision therapies and nanomaterials – using smart materials (like nanoparticles) to target and kill tumor cells more effectively.

The ideal solution would be a single multifunctional material that can:

• Help destroy residual tumor cells after surgery

• At the same time, promote blood vessel growth (angiogenesis)

• And support new bone formation (osteogenesis) to repair the defect

This study aims exactly at that goal.

The Core Idea: A 3D Scaffold Loaded with 2D Nb₂C MXene Nanosheets

The authors combine two advanced material concepts:

1. 3D-printed bioactive glass scaffold (BGS)

   • This is a porous, bone-like structure made of bioactive glass.

   • BGS is already known for:

     • Good biocompatibility (safe for cells and tissues)

     • Good osteoconductivity and osteoinductivity (supports bone formation)

     • A reasonable degradation rate and stable release of its ions

2. 2D Nb₂C MXene nanosheets (NSs)

   • MXenes are a family of 2D materials made from carbides, nitrides, or carbonitrides.

   • Nb₂C MXene is a niobium carbide with a thin, sheet-like structure.

   • It has several important properties:

     • It is biocompatible and biodegradable.

     • It strongly absorbs light in the NIR-II window (1000–1700 nm), especially around 1064 nm.

     • It can convert NIR laser light into heat (photothermal effect).

     • As it degrades, it releases Nb-containing species that can affect biological processes.

The authors integrate these two into one system:

A 3D-printed bioactive glass scaffold coated with ultrathin Nb₂C MXene nanosheets, called NBGS.

This composite scaffold is designed to:

• Allow NIR-II light-triggered photothermal therapy to kill osteosarcoma cells.

• Promote blood vessel formation in the defect site.

• Support new bone formation and scaffold degradation in a coordinated way.

How the MXene and the Scaffold Are Made and Combined

1. Making the Nb₂C MXene Nanosheets

The Nb₂C MXene nanosheets are prepared from a parent material called Nb₂AlC, which is a “MAX phase” ceramic containing niobium (Nb), aluminum (Al), and carbon (C).

The process is as follows (simplified):

• Nb₂AlC ceramic is first made and has a layered structure (seen by SEM).

• The material is then treated with hydrofluoric acid (HF) to selectively remove the Al layer.

• This creates a multilayered Nb₂C MXene, where Nb and C layers remain while Al is largely removed.

• Then, a tetrapropylammonium hydroxide (TPAOH) solution is used as an intercalating agent to help separate the layers further.

• After this, few-layer or single-layer Nb₂C nanosheets are obtained, visible as ultrathin transparent sheets under TEM.

Analytical techniques confirm this transformation:

• Element mapping shows that Al content decreases markedly after HF treatment.

• XPS (X-ray photoelectron spectroscopy) reveals a stronger Nb signal and weaker Al signal in Nb₂C NSs compared with the starting Nb₂AlC.

• Raman spectroscopy shows that characteristic Al-related vibration signals weaken or disappear, while Nb–C related modes remain, proving that the layered structure is retained but the Al layers are removed.

In short, the authors successfully convert Nb₂AlC into ultrathin Nb₂C MXene nanosheets suitable for biological applications.

2. Making the 3D Bioactive Glass Scaffold (BGS) and Coating It

The 3D-printed bioactive glass scaffolds are prepared following their previous work. These scaffolds:

• Have a bone-like interconnected porous structure.

• Are designed to match the mechanical and structural needs of bone tissue.

• Are already known to be bioactive and bone-friendly.

To integrate Nb₂C MXene onto these scaffolds, the authors:

1. Soak the BGS in aqueous Nb₂C NS suspension for 10 minutes at room temperature.

2. Dry the scaffolds at 37 °C for 4 hours.

3. Repeat this soak-and-dry cycle three times.

By changing the concentration of the Nb₂C suspension, they create several versions:

• 0.25 NBGS – coated with 0.25 mg/mL Nb₂C

• 0.5 NBGS – coated with 0.5 mg/mL Nb₂C

• 1.0 NBGS – coated with 1.0 mg/mL Nb₂C

These are later used to evaluate how the amount of MXene influences performance.

Characterization confirms that:

• The scaffold color changes from white to dark as more Nb₂C is added, reflecting MXene coating.

• SEM images show that the 3D porous architecture is preserved after coating.

• With higher Nb₂C loading, the surface becomes more covered and some micropores are partially blocked, but the scaffold structure remains.

• Element mapping shows Nb and Si signals in the same region, confirming that Nb₂C sits on the bioactive glass.

• XRD reveals a new peak around 20° associated with Nb₂C.

• XPS shows a clear Nb 3d peak in NBGS but not in BGS.

• Raman spectra show an additional band around 250 cm⁻¹, consistent with Nb₂C vibrations.

Together, these results show that the Nb₂C nanosheets are successfully integrated onto the 3D bioactive glass scaffold, forming the composite NBGS.

Photothermal Properties: Using NIR-II Light to Kill Osteosarcoma Cells

1. Why NIR-II (1064 nm) Light?

Light in the NIR-II window (1000–1700 nm) has advantages over traditional NIR-I (750–1000 nm):

• Deeper tissue penetration

• Less scattering and better spatial resolution

• More effective and controllable heat generation deeper in tissue

Previous work had already shown that Nb₂C MXene can serve as a photothermal agent to treat tumors (e.g., breast cancer) using NIR-II light, with effective heating up to a few millimeters under the skin.

Here, the same principle is applied to osteosarcoma.

2. In Vitro Photothermal Behavior of NBGS

The authors first expose BGS and different NBGS versions (0.25, 0.5, 1.0) to a 1064 nm laser.

Key observations:

• Temperature rises significantly with NBGS, but not with plain BGS.

• The heating effect is dependent on Nb₂C concentration and laser power density.

• This is tested in both dry (air) and wet (PBS) environments.

As expected, 1.0 NBGS under 1064 nm light shows the strongest photothermal response. This is a solid basis for using it in tumor ablation.

3. Killing Osteosarcoma Cells In Vitro

The authors then test the effect on Saos-2 cells (a human osteosarcoma cell line).

They divide the cells into multiple groups:

• Blank (no scaffold, no laser)

• Laser only

• BGS alone

• BGS + laser

• NBGS alone

• NBGS + laser (NIR-II, 1064 nm, 1.0 W/cm² for 5 min)

Results from CCK-8 cell viability assay:

• Blank, BGS, and NBGS groups maintain >90% cell viability → both BGS and NBGS are non-toxic without laser.

• NBGS + NIR group shows a strong reduction in cell viability (>62% inhibition), indicating effective tumor cell killing due to photothermal heating.

• Other control groups with laser but no MXene do not show significant killing.

Additional tests support this:

• Flow cytometry with Annexin V-FITC/PI shows more than 50% apoptotic cells in NBGS + NIR group, but much lower apoptosis in control groups.

• Live/dead staining (calcein AM and PI) under confocal microscopy shows:

  • Mostly green (live) cells in BGS, BGS + NIR, and NBGS groups without laser-triggered heating.

  • Many red (dead) cells in the NBGS + NIR group, confirming effective photothermal ablation.

These in vitro experiments clearly demonstrate:

NBGS is biocompatible by itself, but becomes a powerful osteosarcoma-killing tool when activated by NIR-II laser light.

In Vivo Tumor Ablation: Photothermal Therapy in Mice

To test the concept in a more realistic setting, the authors set up an ectopic osteosarcoma model in nude mice:

• Saos-2 cells are injected under the skin to form tumors of about 180 mm³.

• Once tumors are established, small incisions are made and either BGS or NBGS scaffolds are implanted into the tumor centers.

The mice are then divided into four groups:

1. BGS

2. BGS + NIR

3. NBGS

4. NBGS + NIR

After 24 hours, NIR-II laser irradiation (1064 nm, 1.0 W/cm², 5 min) is performed on the relevant groups.

Key findings:

• Temperature monitoring shows tumor surface in NBGS + NIR group heats up to ~56 °C, while control groups only reach ~40 °C.

• Histological staining (H&E, TUNEL, Ki-67) of tumors one day later shows:

  • Extensive tumor cell death and nuclear damage in the NBGS + NIR group.

  • Much fewer apoptotic cells and higher proliferation marker (Ki-67) in the control groups.

Safety evaluation:

• Major organs of the mice are examined by H&E staining.

• No significant inflammatory or pathological changes are seen, indicating good biosafety of NBGS and the treatment protocol.

Longer-term outcomes:

• Tumor volume: NBGS + NIR group shows strong tumor growth inhibition, while BGS, BGS + NIR, and NBGS alone show continued tumor growth.

• Body weight: remains stable across all groups, suggesting no major systemic toxicity.

• Survival:

  • BGS: ~20 days

  • BGS + NIR: ~23 days

  • NBGS: ~24 days

  • NBGS + NIR: ~47 days on average

So, photothermal therapy with NBGS plus NIR-II laser irradiation significantly prolongs survival and strongly inhibits osteosarcoma growth in vivo.

Beyond Killing Tumors: Stimulating Blood Vessel Growth (Angiogenesis)

For bone repair, blood vessel formation is crucial. Vessels:

• Deliver oxygen and nutrients

• Bring in bone-forming cells and immune cells

• Help remove waste products

The authors wanted to know whether the presence of Nb₂C MXene in NBGS might positively influence angiogenesis.

1. In Vitro Angiogenesis with HUVECs

They use human umbilical vein endothelial cells (HUVECs) and perform standard assays:

• Scratch (wound-healing) assay – tests cell migration into a gap.

• Transwell migration assay – tests the ability of cells to migrate through a membrane.

• Tube formation assay on Matrigel – tests the ability to form capillary-like tube networks.

When comparing BGS and NBGS:

• Migration of HUVECs is faster and more extensive with NBGS.

• Tube formation is clearly enhanced by NBGS:

  • More branching points

  • Longer tube lengths

Proliferation (measured by CCK-8) is similar between groups, meaning the effect is more on migration and tube formation than on cell growth.

Additionally, gene expression analysis (qPCR) shows that:

• Both BGS and NBGS can upregulate angiogenesis-related genes: VEGF-A, VEGF-B, and FGF2.

• However, NBGS leads to higher and more sustained expression of VEGF-B and FGF2 compared with BGS, especially over 24–48 hours.

In summary:

NBGS significantly improves vasculogenesis and angiogenesis capacity of endothelial cells in vitro compared with BGS.

2. In Vivo Evidence of Neovascularization

To validate this in vivo, the authors implant BGS and NBGS into calvarial defects (skull bone defects) in rats, then perform:

• Microfil perfusion (a radiopaque polymer injected into blood vessels).

• Micro-CT scanning and 3D reconstruction of blood vessels around the scaffolds.

After 3 weeks, 3D images clearly show:

• Denser and richer vascular networks surrounding NBGS compared with BGS.

• Quantitative analysis confirms that new vessel formation is significantly higher with NBGS.

Therefore, Nb-containing MXene in the scaffolds not only enables photothermal function, it also actively stimulates blood vessel development, which is extremely valuable for bone repair.

Supporting Bone Formation: Osteogenesis In Vitro and In Vivo

1. Mineralization Ability and hBMSC Behavior In Vitro

The mineralization potential of the scaffolds is evaluated in simulated body fluid (SBF):

• After soaking, BGS surfaces become rough and are covered by granular mineral deposits with a Ca/P ratio ~1.25.

• NBGS show more deposited minerals and a higher Ca/P ratio (~1.53), closer to that of hydroxyapatite (1.67), the main mineral component of bone.

This suggests NBGS has better bone-like mineral formation capacity, which is beneficial for osteogenesis.

Human bone marrow mesenchymal stem cells (hBMSCs) are then used to test cell–material interactions:

• hBMSCs attach well and spread over both BGS and NBGS, with pseudopods extending into pores → both are cytocompatible.

• Confocal images show that:

  • Cells proliferate over time on both scaffolds.

  • NBGS supports more extensive spreading and larger cell areas, indicating better interaction and possibly stronger signals for osteogenesis.

Osteogenic differentiation is studied via gene expression:

• Key genes measured: COL1, RUNX2, OCN, OPN (all related to bone formation).

• After 3 days, NBGS significantly upregulates COL1, OCN, and OPN compared to BGS, indicating stronger osteoinductive effect.

Alizarin Red S staining after 21 days of culture with osteogenic medium shows:

• More intense calcium deposition in NBGS group than BGS or control, confirming enhanced matrix mineralization.

So, in vitro data clearly show that NBGS promotes osteogenic differentiation and mineralization better than BGS alone.

2. Bone Regeneration in a Rat Calvarial Defect Model

For in vivo bone regeneration, the authors create bilateral skull defects in rats and implant:

• BGS on one side

• NBGS on the other side

They then follow healing over weeks using:

• Micro-CT for 3D evaluation of bone formation

• Dynamic fluorescent labeling (tetracycline, calcein, alizarin red) to track the timing of new bone formation

• Histological staining (H&E, Masson, Goldner trichrome) for detailed tissue analysis

Micro-CT results at 24 weeks show:

• NBGS-treated defects are almost completely filled with new bone tissue, with very little scaffold remaining.

• BGS-treated defects show more residual scaffold and less new bone.

• Quantitative parameters:

  • BV/TV (bone volume / tissue volume) is significantly higher in NBGS group.

  • BMD (bone mineral density) is higher in NBGS-treated areas.

  • Total porosity (TOT) is lower in NBGS group, indicating a denser, more mature bone structure.

Dynamic fluorescence labeling reveals:

• New bone formation happens earlier and more robustly around NBGS.

• Green and red fluorescence (representing later stages of bone formation) are much stronger in NBGS group than in BGS.

Histology confirms these findings:

• NBGS defects show abundant mineralized bone, almost completely filling the defect area, with minimal residual material after 24 weeks.

• BGS defects still show scaffold remnants and less mineralized bone.

• Goldner trichrome staining shows:

  • In BGS group: mixed new osteoid and remaining material.

  • In NBGS group: defect filled almost entirely with green mineralized bone, with no obvious residue of the scaffold.

Importantly, systemic toxicity checks (blood tests and organ histology) show no major abnormalities, confirming that NBGS is biocompatible in vivo.

How Does It All Work Together?

The study suggests that the success of NBGS comes from multiple synergistic effects:

• Photothermal therapy
  Nb₂C MXene converts NIR-II light into heat, killing osteosarcoma cells inside and near the scaffold. This helps control local tumor recurrence.

• Pro-angiogenic effect of niobium species
  As Nb₂C MXene degrades, it releases niobium-containing species that seem to promote blood vessel formation, proven both in vitro (HUVEC assays) and in vivo (micro-CT angiography).

• Pro-osteogenic environment
  The bioactive glass part of the scaffold releases calcium and phosphate ions, which stimulate bone mineralization. The scaffold also offers a 3D porous structure ideal for cell attachment and growth.

• Coupled degradation and bone remodeling
  NBGS degrades at a rate that matches bone formation, leaving space for new bone to grow in. Immune cells and bone-forming cells arrive via the new vessels, speeding up both scaffold degradation and bone regeneration.

In contrast, BGS alone lacks the photothermal function and the niobium-driven pro-angiogenic boost, leading to slower scaffold resorption and less complete bone regeneration.

Final Takeaway

This work shows that Nb₂C MXene–functionalized bioactive glass scaffolds (NBGS) can:

• Efficiently ablate osteosarcoma using NIR-II photothermal therapy, both in vitro and in vivo.

• Promote angiogenesis, creating a rich vascular network around the defect.

• Enhance osteogenesis and mineralization, leading to faster and more complete bone repair.

• Degrade in sync with bone remodeling, leaving behind mature, mineralized bone without long-term residue.

• Maintain good biosafety in animal models.

In other words, NBGS acts as a multifunctional implantable biomaterial that can:

Kill bone tumor cells and guide the regeneration of healthy, vascularized bone at the same time.

This makes Nb₂C MXene–integrated scaffolds a very promising candidate for future clinical strategies in bone tumor therapy and large bone defect repair.