The Significant Impact of the Evolution of Porous Architecture in Sol–Gel Processed-Nanoscale ZnO-Bearing Bioactive Glass in a Physiological Environment

Abstract

Introduction

The incorporation of network stabilizer trace elements such as zinc in bioactive glass is an effective strategy to enhance biological response and modulate degradation kinetics. However, the quantitative relation among ZnO concentration, lattice micro-strain, and evolution of dynamic pore during in vitro mineralization remains unclear.

Methods

Bioactive glass (BG) film with different concentrations of ZnO nanoparticles (NPs) were synthesized through sol–gel spray pyrolysis method at 100 °C, followed by thermal treatment at 250 °C. In vitro bioactivity was assessed by 28 days of immersion in Earle’s Balanced Salt Solution (EBSS). Crystallographic evolution, elemental composition, microstructural morphology, and pore distribution were examined using X-ray diffraction (XRD), Fourier transform infrared (FTIR), field-emission scanning electron microscopy (FESEM), and energy‑dispersive spectroscopy (EDS).

Results

The systematic addition of ZnO NPs controls the matrix dissolution and maintains the integrity of the structure. Quantitative analysis of pore size exhibits large scale degradation of glass matrix in undoped bioactive glass, whereas ZnO NPs embedded BG maintains a controlled micro‑porosity range between 0.4–2.5 μm over 28 days of immersion in EBSS. The value of lattice micro-strain (ε) demonstrates that stability of structure is improved as the concentration of ZnO increases. Furthermore, XRD confirms the progressive nucleation growth of hydroxyapatite (HA) layer, corresponding to characteristic reflection peak at 32.08°. The porous architecture provides high surface area that accelerates rapidly the ion exchange process and nucleation of HA for bone bonding.

Discussion

ZnO NPs act as an effective network modifier that rapidly inhibits the collapse of BG matrix and stabilizes the bioactive interface. The quantitative analysis reveals that micro-strain stability and surface pore dynamics are systematically controlled by ZnO doping, which demonstrates an optimized framework for enhanced bone interfacial bonding and tissue engineering applications.

Introduction

There is significant interest in bio-ceramics for bone repair and reconstruction. Porous bio-ceramics enhances the mechanical strength at the bone-implant interface. Additionally, they facilitate strong surface interaction and provide a larger surface area between tissue and implant.^1,2 When Hench et al³ developed 45S5 bioactive glass (BG) in 1969, the material demonstrated a unique ability to bond with the bone. A few years later, BG was categorized into three types based on their oxide network composition: SiO₂-based, B₂O₃-based, and P₂O₅-based. Among these, silica-based BG was prioritized because it exhibits excellent bioactivity in promoting osseointegration between living bone tissue and implant.^4–6 When the implant is placed in the physiological environment, it forms an HA layer and develops a bioactive attachment with the bone tissue.^7,8 Furthermore, silica-based BGs exhibit high bioactivity, good proliferation, and cell adhesion.⁹ Furthermore, silica-based BGs have several modified chemical compositions, including 58S, 68S, and 77S. Among these, the 58S composition is particularly notable because it exhibits a low tendency for crystallization, while the high content of Ca and P accelerates the rate of HA formation, thereby enhancing cellular mineralization.^10,11

Tetraethyl orthosilicate (TEOS), triethyl phosphate (TEP), calcium nitrate tetrahydrate (Ca(NO₃)₂.4H₂O), and sodium nitrate (NaNO₃) are widely used as standard precursors in sol–gel and melt-derived bioactive glasses. Sol–gel silicate bioactive glasses describe TEOS as the main silica source.^12,13 It is a liquid alkoxide that hydrolyses and condenses to form a silicate network of glass. TEP is introduced as the organic phosphorus precursor, providing phosphate (P₂O₅) to form PO₄^3- groups in glass network. Ca(NO₃)₂·4H₂O is mentioned as calcium source.¹⁴ It dissolves in the solution and during heat treatment the nitrate is removed, leaving Ca²⁺ to enter the glass as a network modifier and to supply calcium for HA formation. NaNO₃ is used as sodium source, where Na⁺ acts as a network modifier that opens the silicate network and increases the reactivity and bioactivity of glass.¹⁵

In biomaterials and optoelectronics applications, silicate-based bioactive glasses bearing with zinc oxide nanoparticles (ZnO NPs) are preferred because of their superior physical, thermal, and mechanical properties.¹⁶ ZnO exhibits unique combination of properties as an intermediate oxide, notably as a glass network former or a network modifier depending on its concentration. This characteristic impacts the glass matrix. When ZnO NPs are incorporated into the silicate network, they mainly act as network modifiers.^17,18 The Zn²⁺ ions break the strong, bridging oxygen Si-O-Si bonds within the long SiO₄ chains. This depolymerization increases the number of non-bridging oxygens (NBOs), which reduces the optical band gap and shifts the absorption edge toward higher energy levels under ultraviolet-visible light.^19,20 In tissue engineering, the structural disturbance caused by these NBO sites lowers the glass softening point and accelerates dissolution kinetics on contact with the physiological fluid. This allows for a rapid therapeutic release of Zn²⁺ ions that simultaneously promotes osteogenesis and provides a strong antibacterial property.^21,22 In summary, ZnO NPs facilitate the formation of bioactive glasses that bond with the bone and are potentially osteogenic and antimicrobial.^23,24

There are several processing methods for BG such as melt-quench, sol–gel, and spray pyrolysis (SP). Bioglass produced via melting is simple and suitable for mass production, but it is difficult to obtain fine particles and nanostructured materials, and also it is fired at a very high temperature (above 1300°C), so they are characterized by a dense structure and small specific surface area, which limits their properties.^25,26 A convenient processing approach is sol–gel. It is chemically versatile, flexible and requires low processing temperatures.^27–29 On the other hand, spray pyrolysis is an alternative and complementary to sol–gel process, which is a low-cost, non-vacuum method for producing powder and film and can be utilized to control the morphology of particles. Film can be deposited on the substrates that are easily adaptable for extensive area deposition and industrial manufacturing operation.^30,31

The objective of this study is to explore and develop a novel, multi-component sol–gel bioactive glass system (ZnO-Na₂O-CaO-P₂O₅-SiO₂) with varying content of zinc oxide nanoparticles (ZnO NPs) in the range of 1–10 wt.%. An accompanying aim is to understand how ZnO act as a structural network modifier to break bridging oxygen bonds and create NBOs. It is demonstrated here that nanoscale structural interference in the glass can be used as a chemical mediator to tune the degradation behavior and surface morphology during 28 days of immersion in biological fluid. By controlling this mediator, the surface topography can be controlled from simple, uniform spherical and rod-like structure toward highly organized, quadrilateral porous architecture. This evolution is beneficial, as it enables the design of an advanced tissue scaffold that maximizes cell attachment and provides controlled release of therapeutic zinc ion. This study systematically correlates quantitative lattice micro-strain stability and pore size distribution with in vitro bioactivity, providing a clear mechanistic layout for bone regeneration.

We processed a series of ZnO-bearing bioactive glasses with enhanced bone regenerative properties and bone-bonding bioactivity.

Experimental

Materials and Methods

The 0.08 M solution of zinc acetate dihydrate (Zn(CH₃COO)₂.2H₂O (99% pure, Rankem LR grade) in isopropyl alcohol (IPA) (99.8% pure, Merck) was used for producing ZnO. To obtain a homogenous solution, the mixture was magnetically stirred for 3 h at room temperature. During this, 0.5 M NaOH aqueous solution was added dropwise until ZnO precipitated. The resulting precipitates were filtered and dried at 50°C for 3 h. To obtain fine particle size, the dry precipitate was milled using a mortar pestle. The resulting powder sample was stored in an air at 400 °C for five hours in a muffle furnace. To further reduce the particle size, the sample was ground again. A pictorial view of the processing method is presented in Figure 1. The structural and morphological confirmation of the synthesized ZnO NPs (XRD, SEM, crystallite size, lattice parameter, bond length, and unit cell volume) is detailed in the supplementary file (Figures S1-S2 and Tables S1-S2).

Figure 1.

Pictorial view of the processing method for ZnO nanoparticles: (A) dissolving zinc acetate dihydrate with IPA, (B) continuous stirring, (C) addition of NaOH as precipitating agent, (D) filtration of precipitate solution, (E) oven drying at 50 °C, (F) crushing the dried precipitate, (G) calcination at 400 °C, and (H) final obtained ZnO powder.

The synthesis of bioactive glass thin film starts with the sol–gel preparation of precursor solution. First, tetraethyl orthosilicate (TEOS) C₈H₂₀O₄Si at 98% (SRL Private Ltd) was dissolved in isopropyl alcohol at 99% (IPA, Loba Chemie) and stirred continuously for 15 minutes to initiate the hydrolysis. Following this, triethyl phosphate (TEP) (C₂H₅O)₃PO at 98% (Otto Kemi Ltd), sodium nitrate (NaNO₃) at 98% (Avarice LR grade), and calcium nitrate tetrahydrate (Ca(NO₃)₂.4H₂O) at 99% (Sigma Aldrich) were sequentially added to the mixture at 15-min interval under continuous stirring to obtain a homogenous distribution of necessary network-modifiers.

To accelerate the hydrolysis and condensation, 1 mL nitric acid (HNO₃) at 25% (Emplura) was introduced as a catalyst. Next, the previously synthesized ZnO nanoparticles of size 30 nm were dispersed into the solution. In this manner, there were six samples synthesized with undoped ZnO, referred to as ZNCPS0. Doping with ZnO at 1 wt.%, 2 wt.%, 5 wt.%, 7 wt.% and 10 wt.% is referred to as ZNCPS1, ZNCPS2, ZNCPS5, ZNCPS7, and ZNCPS10. The resulting mixture was stirred for an additional 2h to obtain complete homogenization and a stable bioactive glass precursor solution. After stirring, the solution was filtered to remove agglomerated particles and ensure a well‑dispersed precursor solution. For thin-film deposition, this filtered solution was loaded into a syringe and atomized onto glass slides held at a substrate temperature of 100°C. Finally, all the coated glass slides were transferred to furnace and dried at 250°C for 2 h to ensure complete solvent evaporation, organic residue removal, and film densification. The bioactive glass film produced with different compositions are listed in Table 1 and the flowchart of ZnO‑doped BG film through the sol–gel process is presented in Figure 2.

Table 1.

Different Compositions of Synthesized Bioglasses

	SiO2	P2O5	CaO	Na2O	ZnO
	(in wt.%)
ZNCPS0	45	7	27	21	0
ZNCPS1	45	7	27	21	1
ZNCPS2	45	7	27	21	2
ZNCPS5	45	7	27	21	5
ZNCPS7	45	7	27	21	7
ZNCPS10	45	7	27	21	10

Figure 2.

Schematic flowchart showing step-by-step process of the BG film formation through sol–gel route.

Characterization: Prior to and After Bioactivity

Cu Kα radiation (λ = 1.5406 Å) at 40 kV was used to record XRD data in continuous mode from 10° to 80° 2θ at a step size of 0.02° and a scan speed of 4°/min at GI angle 0.5° to determine the nature of phase in sol–gel processed bioactive glass. Fourier transform infrared spectroscopy (FTIR) (Bruker Alpha, USA) was used to analyze the different bonds in bioactive glass in the range 2000–500 cm⁻¹ with a resolution of 2 cm⁻¹, in transmission mode. Field-emission scanning electron microscopy (FESEM) (JEOL-7610F) equipped with energy-dispersive spectroscopy (EDS) was used to study the morphology and elemental composition of the synthesized bioactive glass film. All samples were coated with ~10Å thick gold prior to SEM studies. Multiple regions were scanned per sample to confirm uniform surface morphology.

In vitro Bioactivity

To examine the in-vitro bioactivity test of synthesized bioactive glass film, buffered saline solution (EBSS) was used. To conduct bioactivity test, a small part of sprayed film was taken individually from each sample (ZNCPS0, ZNCPS1, ZNCPS2, ZNCPS5, ZNCPS7, and ZNCPS10) and added to 5 mL EBSS in a petri dish and stored in an incubator at 37 °C for 14 and 28 days, respectively. After completion the immersion period, the film was characterized.

Results and Discussion

Figure 3a–f shows the XRD patterns of undoped and ZnO nanoparticles (NPs) bearing (1, 2, 5, 7 and 10 wt.%) bioactive glasses before immersion. The patterns demonstrate that low-angle peaks at 11.4°–12.3° and 15.6°–15.7° arise from diffuse scattering of disordered amorphous glass network and short-range ordering of silicate and phosphate groups.^32,33 The peaks at 20.31°, 23.01° and 24.78° were from the amorphous halo caused by randomly arranged Si-O-Si bonds in the glassy matrix, confirming the amorphous nature of the samples.³⁴ In 1 wt.%, 2 wt.% and 5 wt.% ZnO NPs bearing glasses, no sharp crystalline peaks observed, indicating that ZnO NPs were completely dissolved in the glass network. However, at higher content of ZnO (7 wt.% and 10 wt.%), peaks appear at 31.65°, 34.82° and 36.67°, which correspond to (100), (002) and (101) crystallographic planes of hexagonal wurtzite ZnO,³⁵ matching with JCPDS #36-1451. These peaks corresponding to the hexagonal wurtzite phase of ZnO NPs appear because the solubility limit of ZnO NPs in the glass network is exceeded at higher amount, leading to a separate nanocrystalline ZnO formation. Other weak peaks at 37.6°–50.99° are minor ZnO reflections along with amorphous background.^36,37

Figure 3.

XRD patterns of ZnO NP-bearing bioactive glass samples prior to immersion. Images (a), (b), (c), (d), (e), and (f) correspond to 0, 1, 2, 5, 7, and 10 wt.% of ZnO nanoparticles embedded bioactive glass.

After immersion in physiological fluid, i.e EBSS, all compositions show clear evidence of HA formation, shown in Figures 4a–f and 5a–f. At 14 days, new sharp peaks appear at 32.08°, 45.84°, 66.57° and 75.64°, which respectively correspond to the (211), (222), (211) and (213) crystallographic planes of HA (Ca₅(PO₄)₃OH), presented in Figure 4. The matching JCPDS card number is #09-0432. After 28 days, the apatite reflections at 32.08° (211), 45.84° (222), 66.57° (211) and 75.64° (213) become sharper and more intense for all compositions as shown in Figure 5, demonstrating the growth of a thicker, more crystalline HA layer on the glass surface.³⁸ The key observation here is that, in all compositions, despite the ZnO content, HA formed during immersion, with well-defined apatite peaks after 28 days, confirming that the inclusion of ZnO NPs up to 10 wt.% does not inhibit bioactivity but coexists with the formed HA layer on the glass surface. To have a clarity on the presence of ZnO in hexagonal wurtzite phase, the selected XRD peaks are shown in Figure 6.

Figure 4.

XRD patterns of ZnO NP-bearing bioactive glass samples after 14 days of immersion in EBSS under static condition, where the symbol Φ denotes the initial reflection peaks of crystalline HA layer. Images (a), (b), (c), (d), (e), and (f) correspond to 0, 1, 2, 5, 7, and 10 wt.% of ZnO nanoparticles embedded bioactive glass.

Figure 5.

XRD patterns of ZnO NP-bearing bioactive glass samples after 28 days of immersion in EBSS under static condition, where the symbol Φ denotes the reflection peaks of crystalline HA layer. Images (a), (b), (c), (d), (e), and (f) correspond to 0, 1, 2, 5, 7, and 10 wt.% of ZnO nanoparticles embedded bioactive glass.

Figure 6.

XRD patterns of ZNCPS samples representing a detailed view of specific diffraction peaks of hexagonal wurtzite phases of ZnO nanoparticles.

In ZnO NP-bearing bioactive glasses, Zn²⁺ ions play a dual structural role in the silicate glass network, acting primarily as a network modifier but also as a network former.³⁹ At low to moderate ZnO content (1 wt.%–5 wt.%), Zn²⁺ ions mainly function as a network modifier and they occupy interstitial sites in the silicate network and break Si-O-Si bridges, creating NBOs, according to the reaction as given below:⁴⁰

The breaking of the silicate network increases the number of NBOs and makes the glass more reactive and bioactive. Zn²⁺ has a high field strength similar to Mg²⁺, which increases glass density, refractive index, and activation energy while decreasing thermal expansion. At higher concentration of ZnO NPs, Zn²⁺ enters the glass network in tetrahedral coordination (ZnO₄), forming Zn-O-Si or Zn-O-P linkages and participating in network connectivity.⁴¹ Molecular dynamics studies have shown that Zn²⁺ cations are easily incorporated into the free volume of the silicate network, forming Si-O-Zn bonds and stabilizing the structure. This enables ZnO to enhance bioactivity while maintaining structural stability.⁴²

The HA peak intensity represented in Table 2 demonstrates time- and composition‑dependent mineralization. The intensity increases from day 14 to day 28 at 32.08° in ZNCPS2, ZNCPS5, and ZNCPS10, confirming progressive crystallization and continuous growth of mature HA layer. In contrast, some samples, ZNCPS0, ZNCPS1, and ZNCPS7, exhibit fluctuating or decreased intensities over time because of dynamic dissolution-precipitation process.^43,44

Table 2.

Variation in Characteristic Hydroxyapatite (HAp) Peak Intensities

Samples	Immersion Period	Diffraction Angle of HA Peak
		32.08°	45.84°	66.57°	75.64°
ZNCPS0	14D	45542	2251	3516	–
	28D	10309	7883	927	1024
ZNCPS1	14D	429218	–	46,406	–
	28D	3072	1006	946	–
ZNCPS2	14D	12376	1783	1434	1201
	28D	117695	8039	8640	1765
ZNCPS5	14D	25176	6475	1504	8369
	28D	738912	13,647	60,715	–
ZNCPS7	14D	111774	10,985	7308	4191
	28D	12730	2819	1335	1963
ZNCPS10	14D	22446	5750	–	1109
	28D	82282	26,076	7625	2526

The structural and interfacial stability was analysed by crystallographic micro-strain of HA layer, shown in Table 3. It is observed that the calculated lattice micro-strain value of HA peak remained very low (up to 3 decimal places), confirming the high crystallographic stability and minimum internal stress. In addition, the thin film exhibited strong adherence to the glass substrate because of their similar silicate base. This interfacial bonding mimics the coherent adhesion and mechanical stability when the material comes into contact with natural bone tissue in vivo.^45,46

Table 3.

Crystallographic Micro-Strain (ε) for ZnO Embedded Bioactive Glass Thin Films After Immersion in EBSS

Sample	Immersion Period
	14 days	28 days
ZNCPS0	0.0024	0.0004
ZNCPS1	0.0033	−0.0045
ZNCPS2	0.0031	−0.0004
ZNCPS5	0.0004	−0.0003
ZNCPS7	0.0023	−0.0021
ZNCPS10	0.0005	−0.0023

Figure 7a–c shows FTIR spectra of ZnO NPs bearing bioactive glass. Before immersion, peaks demonstrate the basic structure of bioactive glass, in Figure 7a. The peak at 525 cm⁻¹ arises from bending of Si-O-Si bonds, which is characteristic of a silicate glass network. The band observed at 605 cm⁻¹ is due to bending vibrations of phosphate groups (P-O / PO₄^3-), showing that phosphate is part of glass structure. Peaks at 693 cm⁻¹, 754 cm⁻¹, and 782 cm⁻¹ are all related to bending vibrations of Si-O-Si and O-Si-O in SiO₄ tetrahedra, confirming that the glass has an amorphous silicate framework.⁴⁷ The peak observed at 882 cm⁻¹ and 945 cm⁻¹ are from Si-O⁻ stretching with non-bridging oxygen, meaning some silicate bonds are broken by modifiers (Na⁺, Ca²⁺, Zn²⁺), which makes the glass more reactive. The strong peak at 1025 cm⁻¹ is the asymmetric Si-O-Si stretching band, showing that the silicate network is dominant. The band at 1212 cm⁻¹ comes from asymmetric P-O stretching in phosphate units, confirming that phosphate groups are incorporated into network.⁴⁸ At higher wavenumbers, 1374 cm⁻¹ is related to O-H bending or weak carbonate vibrations, reflecting adsorbed water and early carbonate from air. The peaks at 1446 and 1517 cm⁻¹ are due to C-O stretching in carbonate (CO₃^2-) groups, showing that some carbonate species are already present on glass surface. The band observed at 1737 cm⁻¹ is from C=O stretching of adsorbed carbon‑containing species.⁴⁹

Figure 7.

FTIR spectra of ZnO NP-bearing bioactive glass. Images (a), (b), and (c) correspond to 0, 14, and 28 days of immersion, respectively, and ZNCPS0, ZNCPS1, ZNCPS2, ZNCPS5, ZNCPS7, and ZNCPS10 correspond to 0, 1, 2, 5, 7, and 10 wt.% of ZnO nanoparticles embedded bioactive glass.

After 14 days of immersion, the FTIR spectrum exhibits that the glass surface has started to react and form a new mineral layer, as illustrated in Figure 7b. The peak at 525 cm⁻¹ still represents Si-O-Si bending, meaning the original glass network is still present underneath the surface layer. 583 cm⁻¹ and 607 cm⁻¹ band are assigned to P-O bending in PO₄^3- groups of calcium phosphate or apatite‑like phases, indicating that a phosphate-rich layer is forming.⁵⁰ The peaks at 668 cm⁻¹, 714 cm⁻¹, and 763 cm⁻¹ come from Si-O-Si and O-Si-O related bending in a region that is now affected by the new surface layer, showing structural rearrangement of the near-surface silicate network. The band at 812 cm⁻¹ is related to Si-O or P-O vibrations and appears when a silica-rich or apatite-rich surface layer forms. The peak at 940 cm⁻¹ is associated with Si-O⁻ stretching of N, indicating that the glass network near the surface is still depolymerized and active.⁵¹ The broad absorption band observed near 1000–1100 cm⁻¹ (centered at 1025–1030 cm⁻¹) corresponds to asymmetric stretching of Si-O-Si and non-bridging oxygen (Si-O-NBO) bonds within the amorphous silicate network. The band at 1190 cm⁻¹ is related to P-O stretching in phosphate units. The band observed at 1343 cm⁻¹ corresponds to O-H and carbonate-related vibrations, while 1526 cm⁻¹ is due to C-O stretching in carbonate (CO₃^2-), showing that carbonate is being incorporated into the new calcium phosphate layer.⁵² The band at 1638 cm⁻¹ comes from H-O-H bending of hydrated surface and 1693 cm⁻¹ from C=O stretching of carbon-containing species, indicating a hydrated, carbon‑rich reaction layer on the surface.⁵³

After 28 days of immersion, the spectrum demonstrates that the surface layer has grown and become more mature, shown in Figure 7(c). The peak at 523 cm⁻¹ is still Si-O-Si bending, proving that the original glassy structure remains below the reacted layer. The bands observed at 576 cm⁻¹ and 626 cm⁻¹ are P-O bending vibrations in PO₄^3- groups of apatite, and their presence shows that an apatite layer is formed. The band at 697 cm⁻¹ is related to Si-O-Si/O-Si-O bending. The peak at 810 cm⁻¹ is due to Si-O or P-O vibrations, stable with a developed reaction layer.⁵⁴ The peak at 1001 cm⁻¹ is from P-O stretching in phosphate (PO₄^3-) groups with some contribution from Si-O vibrations, indicating a strong apatite contribution. The band at 1081 cm⁻¹ arises from overlapping asymmetric Si-O-Si and P-O stretching modes, a mixed silica-phosphate surface layer on bioactive glasses. The band at 1215 cm⁻¹ is assigned to asymmetric P-O stretching, showing organized phosphate species. 1367 cm⁻¹ band is associated with O-H bending and carbonate-related vibrations, and 1515 cm⁻¹ with C-O stretching in carbonate (CO₃^2-), both confirming a hydroxy carbonate apatite (HCA) layer containing carbonate.⁵⁵ The band at 1734 cm⁻¹ is from C=O stretching of adsorbed carbon‑containing species. The 28‑day spectrum indicates that ZNCPS develops a hydrated, phosphate- and carbonate‑rich surface layer, consistent with a mature HCA coating on the ZnO‑doped bioactive glass.⁵⁶

SEM analysis was employed to investigate the surface modification of the ZNCPS samples across different immersion periods and magnifications, shown in Figures 8 and 9. Before immersion, ZNCPS0 shows a smooth surface with circular pores surrounded by tiny particles as shown in Figure 8. After immersion of this sample in EBSS for 14 days, it changes into rough surface with larger pores and an accumulated mineral layer at the edges. By day 28, a large dominant pore appears filled with an interconnected mineral layer. As we see the morphology of this sample at higher magnification, it exhibits a rough surface with large pores and small particles dispersed across the surface, as shown in Figure 9. After 14 days immersion, the surface remains rough, but the pores become well-covered with white solid substances, forming eye-shaped structure. By day 28, a large pore appears filled with fluid and interconnected mineral layer. This morphological transition is highly significant because the development of unique, eye-shaped micro-nucleates and interconnected pores increases the active surface area to accelerate bone cell attachment and nutrient transport. In the absence of ZnO NPs, the silica-based glass network dissolves in a specific manner, continuously releasing Ca²⁺ and PO₄^3- ions. This allows a mineral layer to spread uniformly and without interruption. ZNCPS1 exhibits an uneven surface covered with a mix of small and large pores, along with dispersed, accumulated materials before immersion, as shown in Figure 8. After 14 days, the surface becomes smoother as the initial pores fill up, leaving only some small pores visible, with distinctly showcasing an interconnected material framework inside. By day 28, a textured and fully interconnected mineral layer completely covers the surface. At higher magnification, Figure 9 shows a rough surface with dispersed small particles and filled with spherical pore. After 14 days, it shows a mixed rough and smooth texture with pores that are completely filled with a precipitated crystalline HA mineral layer. On day 28, the morphology transformed into a highly rough, deeply textured structure with interconnected mineral layers. This morphological evolution is important for biomedical applications and the formation of a dense, interconnected layer indicative of in vitro bioactivity. A small content of ZnO NPs acts as a network modifier, slowly releasing ions into fluid to induce changes in chemical environment without causing disruption. The small content of ZnO NPs reduces the surface energy, causing the particles to transform in smooth, uniform, spherical shape and coalesce.⁵⁷

Figure 8.

High‑magnification (5k) SEM micrographs of ZnO NP-bearing bioactive glass samples prior to and after 14 and 28 days of immersion in EBSS.

Figure 9.

SEM micrographs (10k magnification) of ZnO NPs‑bearing bioactive glass samples prior to and after 14 and 28 days of immersion in EBSS.

Before immersion, ZNCPS2 has a rough surface on which uneven pores and small particles are visible at low magnification, as shown in Figure 8. After 14 days, it changes into a smoother surface with long, elongated pores that have mineral deposits at their edges and insides. By day 28, a beautiful, interconnected honeycomb-like network covers the surface, scattered with small, newly nucleated mineral particles. Figure 9 shows the higher magnification of ZNCPS2, displaying a smooth surface with partially filled, irregular pores. After 14 days, it maintains smooth texture but forms large pores filled with material that overflows onto the surface. By day 28, the morphology transforms into a rough surface with a honeycomb-like structure and agglomerated particles. This honeycomb shape is highly significant because it perfectly mimics the porous structure of natural bone, which makes it much easier for body fluids to flow through it while the continuous mineral growth confirms long-term bioactivity. At this concentration, the role of ZnO NPs shifts to promoting directional growth. As time progresses, the released ions accumulate and transform the texture from random grains to aligned clusters. This zinc promotes linear, directional expansion of mineral deposit, resulting in an elongated structure. In Figure 8, ZNCPS5 demonstrates a mixed rough and smooth surface with internally filled pores and smaller visible opening before immersion. On day 14, it turns smoother as the main pores get mostly filled, leaving only small empty gaps. By day 28, the smooth surface features filled pores packed with various small openings, creating a pore inside pore alongside scattered particles on the surface. The higher magnification of ZNCPS5, shown in Figure 9, exhibits a mixed smooth and rough surface with clear pore structures before immersion. On day 14, some pores are completely filled, alongside a distinct spherical hump containing visible pores. By day 28, the surface becomes smoother as the number of pores decreases, exhibiting agglomerated surface particles and small square-shaped particles. The reduction in pore count and the appearance of unique square-shaped particles and spherical humps increase surface complexity, which is useful for bone tissue engineering. Here, ZnO NPs act as a moderate kinetics inhibitor. It slows down the initial precipitation process in silica gel, prevents the growth of long, needle-like structure over time, and forces the small particles to pack tightly together, forming a dense structure.²⁴

Before immersion, ZNCPS7 shows a smooth surface with various pores, some of which appear internally filled, as shown in Figure 8. On day 14, a rough surface covered with spherical, agglomerated particles and deposition of small white precipitate is observed. By day 28, very small pores are visible on the rough texture, which are completely filled with small particles, and some particles are settled onto the surface of remaining voids. The higher magnification of ZNCPS7 demonstrates a uniform surface with irregular pores, as shown in Figure 9. After 14 days, it develops a rough texture with an agglomerated layer depositing along the edges of pores. By day 28, the surface converts into smooth as the pore sizes become very small, indicating that the pores have been filled, with small particles placed on top of filled openings. This transition is vital for bone repair. The excess amount of ZnO NPs acts as a factor that disrupts the structure. The high concentration of zinc ions creates chemical saturation in fluid, triggering disordered nucleation. This results in the formation of porous, spherical surface with a highly textured appearance.^58,59 ZNCPS10 exhibits a smooth surface with partially filled, nested pores, as shown in Figure 8. After 14 days, the pores fill with interconnected mineral layers and accumulated particles appear on the surface. By day 28, the pores completely fill, though some empty spaces represent distinct butterfly- and quadrilateral-shaped voids in which mineral agglomerates. At higher magnification, Figure 9, ZNCPS10 exhibits a smooth surface with irregular pores and a nested pore inside pore structure before immersion. After 14 days, it develops large pores filled with fluid. By day 28, surface appears unique quadrilateral pores that are internally filled with fluid. This type of development is vital because distinct geometric growth and filled pore networks increase surface complexity to optimize bone cell attachment. Adding a higher concentration of ZnO NPs reveals a smooth surface and quadrilateral pores, representing an optimal thermodynamic balance. Instead of forming spherical or needle-like shapes, the ions become trapped within a confined space due to high chemical saturation. The voids remaining between these blocks form an interconnected network of open pores.

The morphological evolution is governed by continuous ion exchange and dissolution-precipitation processes in physiological environment. The initial glass network breaks down, releasing ions to form a silica-rich layer where calcium and phosphate rapidly precipitate, filling the pores with interconnected minerals. Over 28 days, specific chemical saturation promotes continuous mineral growth into distinct geometric shapes like butterfly and quadrilateral by controlling crystal expansion to specific pathways.

Role of ZnO Nanoparticles in the Evolution of Spherical Morphology

SEM was used to examine the development, distribution, and structural integrity of the spherical features across the ZnO NPs bearing bioactive glass. After 14 days of immersion in EBSS, SEM image of ZNCPS10 exhibits a significant structural transformation, changing from a flat surface into distinct, well-defined micro-spherical ball, as shown in Figure 10. The image shows that these unique spheres were constructed from flat, rectangular geometric building blocks that pack tightly together.⁶⁰ The surface energy minimization occurred because of incorporation of 10 wt.% of ZnO NPs, which impedes the natural growth of long needles and forces the crystals to bind together into these dense, blocky micro-spherical agglomerates. After 28 days of immersion in EBSS, Figure 10 demonstrates the spherical morphology of samples ZNCPS1 to ZNCPS10. The morphology of ZNCPS1 demonstrates that the film develops dense spherical shape. The low amount of ZnO NPs allows continuous mineral precipitation over the period. That is why the sphere is heavily coated with accumulated, micro-crystalline materials, giving a smoother, cauliflower-like appearance. SEM images of ZNCPS2 show a beautiful micro-spherical cluster. At high magnifications, these spheres appear as long, elongated rod-like structures that grow outward from the center. Because of low concentration of ZnO NPs (2 wt.%), a network distortion occurred in the silica matrix, which altered the chemical environment, directly influencing the material’s surface characteristics and dissolution rate in a more controlled manner and forming these distinct rod-like structures. The morphological analysis of ZNCPS5 demonstrates a well-defined micro-spherical cluster. At high magnification, these spheres were composed of small bullet-like crystalline structures that are closely packed.⁶¹ The presence of 5 wt.% ZnO NPs acts as a growth-limiting factor that inhibits extended crystallization along the axis. Instead of long structures, the material experiences a compact growth producing distinct wood‑gyro shapes that form dense, uniform spheres. ZNCPS7 exhibits a unique micro-spherical morphology. At higher magnification, the surface of sphere was porous, showing an open network and also small clustered mineral particles precipitated and scattered across the surface. The higher content of ZnO NPs (7 wt.%) creates disorder in structural network formation, preventing long-range crystal development, leading to this mixed structure with small, clustered crystalline deposits on a porous surface. The micrograph of ZNCPS10 observed the unique micro-spherical morphology. The spheres exhibit a highly canalicular morphology with a semi-porous, fan-like orientation radiating from the centre. This textured appearance shows visible signs of surface erosion and degradation due to 28-day immersion. Within these capillaries and pathways, small, clustered particles are visible, indicating that secondary mineral precipitation is occurring alongside dissolution process.

Figure 10.

SEM images showing a dense spherical surface morphology of ZnO NPs‑bearing bioactive glass samples after immersion in EBSS.

The development of these spherical morphologies confirms that the material is bioactive and forms an active interface with natural bone and also provides a specific surface area, allowing for a steady and controlled release of essential ions (Zn²⁺ and Ca²⁺), which is expected to destroy the bacterial cell membranes and form biofilm without requiring antibiotics.⁶² When the glass slides are immersed in biological fluid, they begin to dissolve, releasing sodium (Na⁺), calcium (Ca²⁺), and zinc (Zn²⁺) ions. The remaining silica network combines and forms a soft, negatively charged gel layer on the surface. Given that the opposite charges attract, this negative gel pulls the positive calcium and zinc ions along with phosphate fluid (PO₄^3-) ions back down to the surface. This deposition creates a force that promotes the nucleation of small mineral particles.⁶³ To keep their surface energy as low as possible, these new particles grow uniformly outward in all directions, creating three-dimensional shape.⁶⁴ However, the released zinc ions alter the crystal growth kinetics. Instead of forming long and sharp needles, the zinc acts as a growth-limiting factor that promotes the crystals to form compact geometric building blocks. Ultimately, these blocks stack and interlock tightly together to create a dense layer.

These micro-spheres and porous morphology provide significant advantages to biomedical devices and bone tissue engineering. The high surface area of micro-rough, channelled sphere mimics natural bone topology, allowing proteins to adsorb and guiding bone cells to attach and grow much faster to accelerate healing. Additionally, these porous pathways and building blocks act as reservoirs, enabling slow release of therapeutic Zn²⁺ ions to provide long-lasting antibacterial and anti-inflammatory protection.^65,66

Considering the high surface-area-to-volume ratio of ZnO NPs, their chemical reactivity increases on contact with EBSS. Driven by high surface energy, they dissolve rapidly and release Zn²⁺ ions into surrounding environment at a fast and controlled rate. As regards bone tissue engineering, the rapid release of ions is crucial, as an increased amount of zinc ions accelerates bone healing by immediately initiating the process of osteoblast formation.^67,68 Additionally, it exhibits strong antibacterial effects to prevent infection and regulates the inflammatory response.

Figures 11–13 show the energy-dispersive spectroscopy (EDS) of ZnO NPs embedded bioactive glass series, demonstrating the elemental composition before immersion alongside after 14 and 28 days of fluid interaction. EDS analysis confirmed compositional progression of the bioactive glass series, represented in Table 4. Prior to immersion, the elemental spectra verified the presence of matrix constituents, Zn, O, Ca, P, Si, and Na. Following immersion, the appearance of Cl alongside Zn, Na, Ca, P, and Si indicated active chemical interaction with biological fluid. The weight percentage of Zn demonstrated a concentration‑dependent addition across the ZNCPS0, ZNCPS1, ZNCPS2, ZNCPS5, ZNCPS7, and ZNCPS10 samples. Before immersion, the Zn content recorded at 0, 5.7, 2, 6.6, 5.7, and 9.3. After 14 days of immersion, these values shifted to 0, 3.2, 4.2, 7.8, 8.3, and 7.2. By day 28, the Zn values settled at 0, 7, 1.8, 7, 2.3, and 1.3. This subsequent decrease in zinc levels, particularly noticeable in the higher-concentration ZNCPS7 and ZNCPS10 samples, confirms that a thick, newly precipitated mineral layer covered the glass matrix.^69–71

Figure 11.

EDS spectra of ZnO NP-bearing bioactive glass samples prior to immersion in EBSS. Images (a), (b), (c), (d), (e), and (f) correspond to 0, 1, 2, 5, 7, and 10 wt.% of ZnO nanoparticles embedded bioactive glass.

Figure 12.

EDS spectra of ZnO NP-bearing bioactive glass samples after 14 days of immersion in EBSS. Images (a), (b), (c), (d), (e), and (f) correspond to 0, 1, 2, 5, 7, and 10 wt.% of ZnO nanoparticles embedded bioactive glass.

Figure 13.

EDS spectra of ZnO NP-bearing bioactive glass samples after 28 days of immersion in EBSS. Images (a), (b), (c), (d), (e), and (f) correspond to 0, 1, 2, 5, 7, and 10 wt.% of ZnO nanoparticles embedded bioactive glass.

Table 4.

Elemental Composition of Undoped and ZnO NPs Embedded Bioactive Glass Films Before and After Immersion in EBSS

Elements	Sample	Immersion Period			Sample	Immersion Period			Sample	Immersion Period
		0 days	14 days	28 days		0 days	14 days	28 days		0 days	14 days	28 days
O	ZNCPS0	70.9	59.1	55.4	ZNCPS1	56.2	52.9	46.3	ZNCPS2	59.1	53.6	50.4
Na		0.3	2.9	3.5		0	0.1	3.6		0	0.4	2
Si		22.4	29.1	25.6		28.7	37.2	0.8		24.9	34.8	30.9
P		0.5	0.4	2.6		2.9	0.5	5.2		2.7	0.5	0.6
Ca		5.8	2.8	6.5		5	1.4	19.7		6.7	1.1	5.5
Cl			5.7	6.3			4.8	17.5			5.3	8.7
Zn						7.2	3.2	7		6.6	4.2	1.8
O	ZNCPS5	61.5	46.7	50.2	ZNCPS7	62.3	56.6	54.4	ZNCPS10	62	54.3	52.6
Na		0	1.7	2.6		0	0.4	1.7		0	2.1	1.9
Si		25.2	19.4	19.1		23.5	22.7	28.2		22.2	14.5	31.4
P		2	4.8	2.9		2.7	2.1	2.7		2.4	4.5	1.5
Ca		5.5	9.4	7.1		4.6	3.8	6.2		4	9.3	5.2
Cl			10.2	11.1			6	4.4			8.1	6.1
Zn		5.7	7.8	7		7	8.3	2.3		9.3	7.2	1.3

The histogram of pore size distribution demonstrates a clear structure evolution of synthesized bioactive glass prior to and post immersion, shown in Figure 14. Initially, ZNCPS0, ZNCPS1, and ZNCPS2 show pore dimensions between 0.4 and 3.5 μm. After immersion in EBSS, the distribution of pore alters because of mineral precipitation and active dissolution. Samples ZNCPS1 and ZNCPS2 demonstrate pore expansion by day 28, reaching up to 1.5–2.2 μm, whereas higher content of ZnO (ZNCPS5, ZNCPS7, and ZNCPS10) maintains a well-confined pore size distribution within 0.8–2.5 μm. This dynamic shift in architecture confirms sustained micro-porosity, which is crucial for fluid exchange and nucleation of apatite.

Figure 14.

Histograms of pore size distribution of undoped and ZnO‑bearing bioactive glass films before and after immersion in physiological solution for 14 and 28 days, illustrating structural evolution of surface micro-porosity.

The present work emphasizes the need for nanomedicine approaches for healthcare that can transition from conventional processing to digital manufacturing, laying the foundation for precision and futuristic healthcare.^72–74

Conclusions

This study demonstrates a successful way to create advanced bioactive glass films by combining sol–gel chemistry with a spray pyrolysis method. By systematically embedding ZnO NPs to the glass network, we found that zinc acts as a powerful structural switch. It perturbs the silicate network to create NBOs, which control how the glass breaks down and interacts with the biological environment. This change promotes a notable transition in surface appearance. As the ZnO NPs concentration increases, the surface completely changes shape, developing from simple, smooth microspheres and micro-rods to highly unique quadrilateral porous structures. When examined in biological fluid, these changes proved highly effective. Within 14 to 28 days, a new, crystalline bone-like mineral layer (HA) successfully formed across the surface and completely fill the open pores. The lower micro-strain value of HA peaks confirms the high crystallographic stability and minimum internal stress. This ability to develop bone-like minerals with releasing zinc ions makes these glass films an effective material for bone repair and biomedical implants.

Disclosure

The authors declare no competing interests.