Introduction

In the last decades, the field of biomaterials has grown at an incredible rate, leading to the development of bioactive materials, which may elicit specific and predictable responses from cells and tissues1. The discovery of Hench glass in 1969 constituted for the first time a revolution in the history of biomaterials2,3. The mechanical, bioactive, and structural properties of the bioactive glasses are largely based on synthesis procedures, composition, particle size, crystallization, etc. Bioactive glasses are made using several approaches. The most common method for making bioactive glasses is the traditional melt-quenching method, in which all the components are well mixed in a ball mill before being melted at an elevated temperature4. In the melt-quenching process, a volatile part such as B2O3 gets evaporated out5,6,7.

In constructing a bioactive glass, understanding how the physicochemical structures of these materials influence their characteristics is critical, as it allows the material to be adapted for particular applications. Each component influences the bioactive glass's performance. Calcium, for example, promotes osteoblast development and apatite layer precipitation8,9. Na2O. K2O. MgO. CaO. P2O5-based glasses found to constitute a promising material for bioactive applications such as bone repair, tissue regeneration in the human body, etc.10,11,12,13. Borate glasses quickly release significant amounts of boron, leading to a high concentration of local boron near the glass. As a result, compared to silicate glasses, the degradation and sintering behavior of borate/borosilicate glass is more controllable14,15,16. In borate glasses, the boron oxide appears in BO3 and BO4 in a network structure that forms 'super structural' units (pentaborate, boroxol ring, diborate, or tetraborate groups), depending on the composition and the kind of added glass modifiers17,18,19,20.

The glass exhibits nonlinear change in its physical properties when one alkali ion is replaced with another alkali content at a constant amount, resulting in a mixed alkali effect (MAE)21. Incorporation of zinc oxide in glass structures is expected to acts as a intermediate oxide either as network former or as network modifier22,23. Glass composition is modified by introducing the ‘dopants’ to the glass network to form the desired glass where it can be bioactive, bioresorbable, and/or biodegradable. Dopants like Cu, Zn, In, Ba, La, Y, Fe, Cr, and Sr as ions lead to trigger the properties. ZnO/MgO additives have been shown to stimulate osteoblast proliferation, differentiation, and bone mineralization24,25,26,27. Zn is an essential trace element that is used by various metalloenzymes for structure, catalysis, or regulatory functions. Zinc is involved in bone metabolism, enhancing osteoblastic bone formation and preventing osteoclastic bone resorption, raising bone mass28. Nutritional zinc supplementation has been demonstrated to have preventive and therapeutic effects on bone loss induced by bone disorders29,30. Investigations have also demonstrated that small quantities of Zn induced early cell proliferation and enhanced differentiation of in vitro biocompatibility studies31,32,33. Methodologies like MTT and MTS are used to assess the viability and cytotoxicity of the glass samples because these methods generally measure cytotoxicity for bulk constructions indirectly by extracting the materials.

According to Saranti et al., boron oxide has a catalytic action that promotes bioactivity34. Neáková et al. developed mesoporous bioactive glass nanoparticles (MBGNs) based on the SiO2–CaO system using a micro-emulsion supported sol–gel method. Zn2+ ions were doped into MBGNs with 8 Mol% ZnO concentration (Zn-MBGNs). The findings investigated that the addition of zinc precursors had no effect on particle morphology, but enhanced their specific surface area when compared to MBGNs35. Lee et al. reported that bone implant and osteointegration utilizing a B2O3-based glass technology represents no toxicity. Bone implant and osteointegration using B2O3 based glass system are reported by Lee et al. without any toxicity36. Kolavekar et al. studied the optical properties of Pr2O3-doped multi-component borate glasses37, TeO2-doped lead borate glasses38, Li+ Ions doped zinc borate glass39 and Er3+ and Er3+/Yb3+ co-doped heavy metal borate glasses40 and showed the effect of the dopants on the photophysical properties of the borate glass.

Bioactive glasses are effective biomaterials for promoting angiogenesis in both hard and soft tissue engineering applications. Metallic ions like Cu2+, Ag2+, Mg2+, Zn2+, Fe3+, Sr2+, and Co2+ have been utilized as dopants in oxide glasses41,42,43. The presence of these ions in the glass network causes antibacterial agents, osteogenesis motivation factors, and angiogenesis enhancers44,45.

The effect of laser irradiation was studied for borosilicate glasses attracted much of scientisits attention because it can stimulate various microspheres that can be controlled within transparent materials due to non-linear optical absorption46,47. The novelty in the work is to study the effect of laser irradiation on the structure and optical properties of ZnO doped borate bioactive glass.

This study is aimed to investigate the effect of ZnO on the structure of bioactive borate glass using X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The effects of laser irradiation on the selected glasses are studied in terms of their UV–Vis absorption spectra and XRD pattern. The cytotoxicity of ZnO-doped bioactive borate glass on cell viability is assessed in a contest to bioapplications.

Experimental work

Materials (glass preparation)

The glasses of compositions 6Na2O + 12K2O. + 5MgO + 20CaO + 4P2O5 + (53-x) B2O3 + xZnO (0 ≤ x ≤ 0.6 mol%), were prepared via traditional melt quenching method. Highly pure chemicals of orthoboric acid (a source for B2O3) and ammonium dihydrogen phosphate (a source for P2O5) supplied by Sigma Aldrich Co were used. Na2O, K2O, MgO, and CaO were added as carbonates provided by El Nasr Pharmaceuticals. All the chemicals were used as received. The chosen glass batches were mixed and melted in an electrical furnace at 1100–1150 °C and swirled to assure homogeneity, and then the melts were quenched and pressed between two steel plates at room temperature to obtain the glasses. The so-obtained glasses were characterized using XRD patterns, FTIR, and UV–visible absorption spectra. Images of the formed glasses are listed in the Table 1.

Table 1 Photo images of borate glass doped with different amounts of ZnO.
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Characterization and analysis techniques

The various methods of analysis and typical settings that describe ZnO doped borate glass are recorded by utilizing the following instruments: (a) the X-ray diffraction pattern (XRD) was recorded in the range of 4° ≤ 2θ ≤ 70° using a Rigaku X-ray diffractometer ultima IV with CuKα radiation of wavelength λ = 0.154600 nm and steps of 0.02°. (b) Microstructure of the samples was examined using a scanning electron microscope (JEOL JSM-6510LV, USA), which used a focused electron beam (operating at 20 kV accelerating voltage) with a magnification up to 40,000 X, where the samples were coated with gold so the surface becomes conducting to measure the images. (c) A FTIR spectrometer (type Nicolet i10, Thermo Fisher Co.) was used to record FTIR spectra of the glasses over a 4000–400 cm−1 range, with a 2 cm−1 step resolution. Measurements were performed on powders dispersed in KBr in a 1:100 ratio in the form of thin pellets. (d) UV/Visible absorption spectra were recorded (in a range of 190 to 1100 nm) for the polished `samples, using a double beam (JASCO V570 UV/Vis./NIR) spectrophotometer, with air as a reference sample. All the glass samples were irradiated for 30 min by a laser beam (λ = 375 nm) with a 150 mV power.

The technique of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay technique was used to assess cytotoxicity and proliferation was purchased from (Serva, Germany). Cells were seeded in well plates and cultured for 48 h to determine the IC50 for the different glasses and compared with the control sample (normal cell without glass). The glass was solubilized in dimethyl sulfoxide (DMSO) stoke before treatment. The reduction in cell growth was measured at (570 nm) (BioTek, Elx800, US) and the results were calculated as a percentage of control. Prism software was used to calculate the IC50 of glass concentrations as well as cell viability.

Results and discussion

XRD patterns and microstructure

Figure 1 presents XRD patterns of the ZnO doped borate glass, which reveal an amorphous structure for all the samples with a broad diffraction peak at 2θ = 29° of diffraction angle ‘2θ’, with no sharp peak in absence of any crystallites. The absence of clearly defined diffraction peaks confirms the samples' glassy nature and rules out the possibility of long-range atomic organization48,49. The absence of Bragg's peak and the amorphous glassy nature of all the glass samples were confirmed by X-ray diffraction patterns due to the presence of potassium and magnesium, in particular, which enhanced glass processability, making it easier to make glass without crystallization and use it in coatings, fibers, scaffolding and other applications50.

Figure 1
figure 1

XRD patterns of different concentrations of ZnO doped borate glass in the range of 4° ≤ 2θ ≤ 70°.

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Figure 2a, b represent SEM of borate glass doped with 0.6 mol% ZnO of lower and higher magnification which showed dispersed particles or grain-like structure appeared in the morphology of 0.6Mol% ZnO doped borate glass. SEM images indicate the domination of the amorphous structure of the borate matrix as a continuous phase. A good agreement with the suggested amorphous structure from the XRD pattern was demonstrated from the SEM images.

Figure 2
figure 2

SEM images of borate glass doped with 0.6 mol% ZnO; (a) lower (b) higher magnifications.

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Figure 3 shows FTIR spectra of the ZnO doped borate glass where there were broad absorption bands that indicate the groups inside the network system. The interpretation of IR of borate glass is summarized as follows: The first broad band around 1396 cm−1 indicated for B–O asymmetric stretching vibration band of trigonal BO3. The second band at 1008 cm−1 pointed to the B–O bond stretching of the tetrahedral BO4. The third band positioned nearly at 714 cm−1 refers to the bending of B–O–B in trigonal BO3 units and the last band at 563 cm−1 points to the vibration of metal cations like ZnO. The bands face no changes when ZnO nanoparticles are added to the composition. The strong appearance of vibrational broad bands of triangular and tetrahedral for borate glass owing to the presence of the two alkali metals Na2O and K2O. Also, many little changes were investigated after the minor addition of ZnO nanoparticles which indicated the effect of the concentration of ZnO nanoparticles on the transformation process. The band appears in the far infrared region 425 cm−1 due to the stretching vibration of transition metal ions such as (Zn2+, and Mn2+). The other main vibrational modes mentioned before are due to the borate glass matrix51.

Figure 3
figure 3

FTIR spectra of borate glasses doped with different concentrations of ZnO.

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PeakFit4.12 computer program was used for the mathematical deconvoluted analysis technique (DAT) that was used to examine and analyze the collected FTIR spectral data of ZnO-doped borate glasses to obtain quantitative information about the internal changes inside the glass matrix. In the range of 1750–500 cm−1, the smeared overlapping bands of triangular and tetrahedral borate groups were resolved, while the region of 3200–3600 cm−1 reveals the vibrational modes of the OH group. The deconvoluted analysis was established using a small number of bands to resolve the spectra and then weaker bands were inserted to improve the fit. Figure 4a–e represents the deconvoluted analysis of ZnO-doped borate glasses and their residuals. The deconvolution process is based on the previous knowledge of the wavenumber of suggested vibrational groups and the second derivative of the spectrum that identifies the accurate position of peak maxima as previously described by different authors52,53,54,55,56. In such cases, the differences between the deconvoluted and measured spectrum can be minimized and plotted as shown in the residual curves.

Figure 4
figure 4

The deconvolution of FTIR spectra with its residual of ZnO doped borate glasses.

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Obtained N4 data of coordinated boron (N4 = BO4/(BO4 + BO3)) for ZnO doped borate glasses is represented in Fig. 5, which indicates boron atoms transformations occur inside the glass matrix after ZnO nanoparticles addition. It can be figured that the value of N4 increased by increasing the concentration of ZnO nanoparticles to 0.2 mol% and then faces two stages of decreasing and increasing. The creation of non-bridging oxygen NBOs can be correlated with the decrease in the N4 ratio with increasing in ZnO nanoparticles content in addition to other parameters. The negative charge on NBOs makes it easier for electrons to be excited at higher wavelengths57. Both ZnO and CaO act to reduce the BO4/(BO4:BO3) ratio and enhanced the glass network by increasing the number of non-bridging oxygen atoms58. In the glass structure, ZnO acts as a intermediate oxide either as network former or as network modifier59, where ZnO nanoparticles is a promising candidate as a modifier with a large band gap, allowing it to be used as a potential optical material48,60. As there was an unnoticeable change ZnO nanoparticles act as a modifier not as a former.

Figure 5
figure 5

Variation of the fraction of four coordinated boron as a function of ZnO nanoparticles concentration.

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Electronic absorption spectra

Ultraviolet–visible absorption spectroscopy represents the optical properties that give information about the electronic structure of the material61,62. UV–Vis. absorption spectra of ZnO doped borate glasses were explained in Fig. 6 in the wavelength range of 190–1100 nm. It can be figured that the absorption of UV–Vis arises from the higher wavelength to the lower wavelength of the glass materials. A high absorption band at the wavelength of 261.75 nm in the UV region of the spectrum was figured out for all doped glasses, which results from the unavoidable trace iron impurities in the raw materials during the glass formation63. While the broad absorption band between 200 and 340 nm is due to high valence or tetrahedral coordination of the transition metal ions in the alkali borate glass which agreed with the data reported in64,65. The nonlinear behavior of the position of the edge at around 340 nm was found to be compatible with the N4 values represented in Fig. 5. The UV absorption of glasses is considered to be influenced by both internal and external factors such as the electronic transitions, which are primarily caused by the addition of dopants and are influenced by the glass structure and chemical bonding66. Also, the change in the position of the absorption edge is due to the variation of oxygen bonding in the glass network67. The addition of metal cations such as Pb, Zn, Cd, and others affects the network formation of B2O3 and SiO4. These additives also act as a network modifier and a nucleating agent for glass crystallization. As a result, the optical properties of borate glasses have changed significantly68.

Figure 6
figure 6

Absorption Spectra of ZnO doped borate glasses in the range of 190–1100 nm.

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Using a diode laser with a wavelength of 375 nm and 150 mW power the ZnO-doped borate glasses were irradiated for 30 min at room temperature. Figure 7a–f represents the absorption spectra for ZnO-doped borate glasses before and after the irradiation process. It can be figured that there was an absorption band in the UV region and no visible bands were observed for ZnO-doped borate glasses before and after the irradiation process as can be seen in Fig. 7a–f, only a change in the absorption intensity for the absorption band in the UV region and a small shift to the edge wavelength λedge were observed. The values of the λedge and direct optical energy gaps were calculated using Tauc plots and the Mott-Davis model \({\left(\alpha h\upsilon \right)}^{2}=B\left(h\upsilon -{E}_{g}\right)\)69,70 are listed in Table 2.

Figure 7
figure 7

UV/vis absorption spectra of ZnO doped borate glasses before and after 30 min of laser irradiation.

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Table 2 Optical energy gap before and after laser irradiation for ZnO doped borate glasses.
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XRD was performed for o.1mol% ZnO doped sample and it’s obvious that clearly defined diffraction peaks were absent confirming the samples' glassy nature and ruling out the possibility of long-range atomic organization48. Figure 8 showed that before and after laser irradiation as there were two broad humps after laser irradiation around 29° and 46° were detected such humps distinguish non-crystalline solids (amorphous solids). Therefore, it can be stated that all studied samples are short-range order solids (glass solids).

Figure 8
figure 8

X-ray diffraction pattern of borate glass doped with o.1 mol% ZnO before and after 30 min of laser irradiation.

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Cytotoxicity assay

It is necessary to evaluate the cytotoxicity of the material used in bio-applications. The culture of normal human skin fibroblasts (HSF) was used and investigated the influence of the glass with different concentrations on these cells.

Glass efficiency and potency in cells exposed to a drug (glass) are commonly evaluated using drug (glass) dose–response assays (e.g. MTT assay), and then the IC50 (half maximum inhibitory concentration) is estimated. With cell-based cytotoxicity studies, the 50 percent inhibitory concentration (IC50) is commonly employed to measure drug potency71. Zinc ions have also been linked to a variety of physiological activities, such as cell proliferation72. The result of cell viability was determined for the undoped and 0.6 mol% ZnO doped borate glasses Table 3. From the data in the table starting from 50 to 1.5625 μM of the glass material where a low concentration of each glass (1.5625 μM) gives a high percentage of viable cells, and also, they are non-toxic. After increasing the concentration of the glass material, the values of cell viability are still good compared to cell viability values in73,74.

Table 3 Values of cell viability of the undoped and 0.6 mol% ZnO doped borate glasses with different concentrations of each glass.
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Figure 9a,b represent the dose–response curves cytotoxicity assay of the undoped and 0.6 mol% ZnO doped borate glasses with different concentrations incubated in cell culture and showed the half-maximal inhibitory concentration (IC50). The standard error of the mean is represented by the error bars. Prasad S75, investigated in vitro cell proliferation using the MTT test on the base glass (BG0B) selected from the SiO2–Na2O–CaO–P2O5 system (S53P4 glass), as well as various modified glass compositions generated by replacing SiO2 in the base glass composition by B2O3 (BG1B, BG2B, and BG3B). It demonstrates that the cell proliferation was better on the B2O3-modified glasses (BG1B, BG2B, and BG3B) compared to the base glass (BG0B). It’s founded that cell growth was better on the B2O3-modified glasses (BG1B, BG2B, and BG3B) when compared to the base glass (BG0B). According to Balasubramanian et al.76 adding boron to bioactive glasses in various quantities has substantial effects on glass structure, glass processing characteristics, biodegradability, biocompatibility, bioactivity, and cytotoxicity. For bone and soft tissue engineering, various compositions of boron-doped, borosilicate, and borate glasses, are being studied. After cytotoxicity tests, the optical microscope image of the cells was taken (Fig. 10a–c). The morphology of cells that extend over the dish surface was not changed in the samples that showed low cytotoxicity as compared to the control sample. The morphology of the cells was approximately similar compared to the control sample, where the pulp cells indicated the survival cells and the rounded or shrunk cells indicated dead cells. It’s concluded that non-toxic behavior is exhibited by the prepared glasses compared to the control sample cells. Also, it is appropriate to be used for human tissue with no harmful effects.

Figure 9
figure 9

Effect of different amounts of (a) undoped glass, (b) 0.6 mol% ZnO doped borate glass on cell viability assay as determined from the absorbance at 570 nm and corresponding IC50 values.

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Figure 10
figure 10

The optical microscope image of (a) undoped glass, (b) 0.6 mol% ZnO doped borate glass after cytotoxicity tests compared to (c) control sample.

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Conclusions

In this study, ZnO doped borate glasses with a composition of 6Na2O + 12K2O. + 5MgO + 20CaO + 4P2O5 + (53-x) B2O3 + xZnO, (0 ≤ x ≤ 0.6 mol%) were synthesized by traditional melt quenching technique. XRD study showed a high degree of amorphous structure for all samples. The formation of borate glass and the interaction with ZnO nanoparticles were indicated successfully by FTIR spectroscopy. Deconvolution analyses were applied to analyze the collected FTIR spectral data and showed a slight change in N4 coordinated boron but wasn’t noticeable due to the minor addition of zinc oxide. The incorporation of TMI was found to produce BO3 and BO4 structural units by shattering the boroxol (B3O6) ring, according to Fourier transform infrared (FTIR) spectra. UV–Vis optical properties were applied, and the optical energy gap was found to be around 3.4 eV. A highly intense band in the UV region in the range between 200 and 270 nm was noticed and found to be due to unavoidable trace elements introduced by raw materials. After the laser irradiation process, the optical energy gap was nearly similar for all samples but there was a change in the absorption intensity. The XRD pattern showed a change in the structure after the irradiation process, which indicate the short-range order of the investigated glass. Good cell viability was found for 0.6 mol% ZnO doped borate glass compared to the undoped glass after using the MTT assay method, the value of IC50 was decreased from 411.9 μM for borate glass to 126.4 μM for 0.6 mol% ZnO doped borate glass. As a result of that, and after future study on biodegradation and activity, ZnO-doped borate glasses with nominal composition is recommended for in vitro and in vivo bio applications.