Comparative in vitro Cytotoxicity and Antioxidant Activity of Biologically Derived Nanohydroxyapatite and Commercial Nanohydroxyapatite

In the sphere of biomedical research, creating innovative biomaterials is vital for addressing challenging healthcare issues. Hydroxyapatite (HAp) is one such biomaterial that has drawn much attention because of its remarkable properties, particularly in regard to hard tissue implants. The different components of HAp as biomaterials are explained in this introduction, along with the intricacies of oxidative stress and its effects during bone implantation, the importance of antioxidants and HAp limits. In addition to outlining the goals and possible contributions of this research, it highlights a novel metal doping method to increase HAp performance.
       
A common biomaterial for hard tissue implants is HAp, a calcium phosphate crystal with the chemical formula Ca₁₀(PO₄)6(OH)₂ (Gülçin et al., 2010). It is highly biocompatible and biodegradable because of its crystalline structure, which resembles the mineral makeup of bone tissue. Because of these intrinsic properties, HAp is an ideal choice for orthopedic and dental applications, where it can be used as a base for dental restorations, coatings for orthopedic implants and bone transplants.
       
As an alternative to bone transplantation, HAp has become more significant in orthopedics because of its capacity to promote osseointegration and bone regeneration. HAp enhances the compatibility of orthopedic implants with host bone tissue when used as a covering material, which promotes recovery. HAp is utilized in dentistry for restorative procedures and dental implants since it is both biocompatible and aesthetically similar to human teeth. HAp has also shown promise in drug delivery systems, where it functions as a carrier for pharmaceuticals, guaranteeing regulated release for better therapeutic results. Oxidative stress is a fundamental physiological condition that results from an imbalance between the body’s defense mechanisms and the generation of reactive oxygen species (ROS) (Schieber et al., 2014).
       
ROS include a variety of species, such as hydrogen peroxide (H2O2), hydroxyl radicals (•OH) and superoxide anions (O2•-). Although ROS are necessary for regular physiological functions, excess ROS can result in oxidative stress, which can harm cells and cause a variety of diseases (Barnes, 2013; Kramer et al., 2014; Machado et al., 2012).
       
Because ROS are produced after bone implantation as part of the body’s immune response and wound healing processes (Mahanty et al., 2023), serve a dual function in tissue repair and can cause oxidative stress and inflammation if their levels are not properly regulated (Jiang et al., 2002), it is crucial to comprehend  how ROS and bone implantation interact in order to improve implant outcomes. Implant-related oxidative stress can result in oxidative damage, which can affect the success of the implant and the healing process (Krishani et al., 2023).
       
The body uses antioxidants as a natural defense mechanism against oxidative stress (Taladrid et al., 2023). By providing electrons to ROS, they neutralize them and stop oxidative damage to biological components. Enzymatic antioxidants such as catalase and superoxide dismutase work in tandem with nonenzymatic antioxidants such as glutathione, polyphenols and vitamins C and E to preserve redox balance (Poljsak et al., 2013) (Hasanuzzaman et al., 2020).
       
To combat the oxidative stress caused by the implantation procedure and subsequent tissue healing, antioxidants are essential during bone implantation. The importance of antioxidant techniques in implantology is highlighted by the fact that imbalances in the antioxidant defense system can lead to increased ROS levels and compromised healing. HAp has limitations in regard to hard tissue implants, despite its clear advantages. Its low mechanical strength is a major drawback that restricts its applicability in load-bearing applications. Additionally, over time, HAp brittleness may result in wear or fracture. Additionally, because ROS generation is elevated during implantation procedures, HAp is unable to effectively counteract oxidative stress, which is a significant issue. The antioxidant activity of nHAp can be evaluated via the DPPH radical scavenging assay, which provides insight into its potential to neutralize free radicals. Building on this concept, we investigated the biocompatibility and osteogenic potential of nHAps using the MG-63 human osteoblast-like cell line (Kumar et al., 2024).
       
A popular human osteoblast-like cell model, the MG-63 cell line, is frequently used to study the activity of bone cells and their interactions with biomaterials. Nanohydroxyapatite (nHAp) is commonly used in combination with MG-63 cells to evaluate its osteogenic potential and biocompatibility. By using an in vitro method, scientists can clarify how nanosized hydroxyapatite interacts with bone cells, offering important new information on its possible use in bone tissue engineering (Acharya et al., 2022).
       
In this study, we performed a DPPH radical scavenging assay and an MTT assay for three samples of nano-hydroxyapatite. The first sample was synthesized from Indian hen eggshell (IE1), the second was synthesized from hybrid or broiler hen eggshell (HE1) via the precipitation method and the third sample was the reference sample nano-Xim HAp 202 (a gift sample from Fliidinova, Portugal).
               
In this study, we performed a DPPH radical scavenging assay and an MTT assay for three samples of nanohydroxyapatite. The first sample was synthesized from Indian hen eggshell (IE1), the second was synthesized from hybrid or broiler hen eggshell (HE1) via the precipitation method described in the article (Patil et al., 2025), and the third sample was the reference sample nano-Xim HAp 202 (a gift sample from Fliidinova, Portugal).

Materials
 
2,2-diphenyl-1-picrylhydrazyl (DPPH) Dimethyl Sulfoxide (DMSO), Sabouraud dextrose agar Plate-(SDA) (Sisco Research Laboratories Pvt. Ltd. (SRL)-India), Methanol  and ascorbic acid (S D fine-chem limited), MG63 (Procured from NCCS Pune) cell line,Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) (Purches from HIMEDIA). (SRL Chem, Cat no.-19427) plates. Fungal Culture (C. albicans, MTCC 854) Procured from Microbial Type Culture Collection and Gene Bank (MTCC)- Chandigarh and Amphotericin B-(Amphocare) The study  of sample preparation of n-HAp was conducted in Pharmaceutical Department of Satara college of pharmacy satara, Maharashtra and study conducted from November 2022 to January 2025.
 
2,2-Diphenyl-1-picrylhydrazyl assay
 
In a 96-well plate, 0.1 ml of 0.1 mM DPPH solution was mixed with 5 μl of a different stock of the test chemical (as stated in Table 1). The reaction was set up in quadruplicate and duplicate blanks were made with 0.2 ml of methanol and 10 μl of standard/sample at various concentrations (Fig 1 and Table 1). Wells without reagents (DPPH) were referred to as blanks, while wells without treatment were referred to as controls (Zero-concentration samples). The plate was left in the dark for half an hour. A microplate reader (μMark, Bio-Rad) was used to measure decolorization at  517 nm at the final stage of incubation. A total of 20 μl of deionized water was added to the reaction mixture as a control. The formula was used to determine the percentage of radical scavenging activity (RSA).

 

Abs sample = Absorbance of sample.
Abs control = Absorbance of control.
       
(Shi et al., 2023; Abdulkadir et al., 2020; Singh et al., 2024). Compared with that of the control, the scavenging activity was displayed as “% inhibition”. IC₅₀ was computed with graph pad prism 6 (software). A graph was prepared between the X-axis (sample concentration) and the Y-axis (% inhibition with respect to the control).
 
Cell sensitivity assay
 
An MTT assay was used to assess the cytotoxicity of the samples to the MG63 cell line. For  24 hours, the cells (10000 cells/well) were cultivated in 96-well plates at 37^oC with 5% CO₂ in DMEM supplemented with 20% FBS and 1% antibiotic solution. The cells were treated with varying quantities the next day (concentrations are specified in Table 4). The cells without MTT were referred to as the blank and the untreated cells were referred to as the control. After a 24-h incubation period, the cell culture was supplemented with MTT solution and incubated for an additional 2 h. After the culture supernatant was removed at the end of the experiment, the cell layer matrix was dissolved in 100 µl of DMSO and the absorbance was measured at 540 nm (Imam et al., 2011) (Morgan et al., 2003). An ELISA plate reader (iMark, Bio-Rad, USA) was used. Images were taken with an AmScope digital camera (10 MP Aptima CMOS) under an inverted microscope (Olympus ek2). The mean ± SEM (standard error of the mean) was used to represent the 50% inhibitory concentration (IC₅₀). To calculate the percentage of viable cells in the MTT assay, we used one formula. In the MTT assay, we used the formula to determine the percentage of live cells (Van et al., 2011; Fotakis et al., 2005; Tihauan et al., 2020; Ngurthankhumi et al., 2024).

 

 

A_test = Test sample’s absorbance.
A_Control = Control sample’s absorbance.

2,2-Diphenyl-1-picrylhydrazyl assay (DPPH assay)
 
The antioxidant activity (DPPH assay) of the samples was calculated on the basis of the experimental results and the 50% inhibitory concentration (IC₅₀). Compared with standard ascorbic acid (Table 3), 50% of the inhibitory concentration was above the dosage limit, i.e., 1000 μg/ml. It was discovered that 3.125 μg/ml ascorbic acid was equal to 250 μg/ml ascorbic acid in sample IE1. Similarly, 50 μg/ml HE1 was comparable to 1.56 μg/ml ascorbic acid, while 1 μg/ml reference sample was identified as being equivalent to 0.78 μg/ml ascorbic acid (Table 3).




       
The results of the DPPH assay suggest that the HAp samples from Indian hen eggshells and hybrid hen eggshells have antioxidant activity comparable with that of the reference sample, which may be attributed to the presence of bioactive compounds such as proteins and minerals. The reference HAp sample presented relatively low antioxidant activity, which may be due to the absence of these bioactive compounds.
 
Cell sensitivity assay (MTT assay)
 
On the basis of the MTT assay results, the negligible cytotoxic activity of samples IE 1, HE1 and Reference was estimated when the MG63 cell line was exposed to varying concentrations of the samples (Fig 2). The results revealed that the IC₅₀ values of the HAp samples were significantly different. The IE1 sample has an IC₅₀ value of 1797 μg/mL, the HE1 sample has an IC₅₀ value of 1348 μg/mL and the reference HAp sample has an IC₅₀ value of 1972 μg/mL, indicating that all the samples have lower cytotoxicity (Table 6), may be more biocompatible and suitable for biomedical applications.






       
This study successfully synthesized and characterized nanohydroxyapatite (nHAp) from Indian and hybrid hen eggshells, evaluating its antioxidant activity and cytotoxicity in comparison to a commercial reference sample. The results indicate that biologically derived nHAp holds great promise as a biocompatible and sustainable biomaterial for various biomedical applications.The DPPH assay revealed that the biologically derived nHAp samples exhibited significant free radical scavenging activity, comparable to the commercial reference. This antioxidant potential may be attributed to the presence of bioactive components such as trace proteins, calcium and phosphate ions, which may enhance radical scavenging. Given that oxidative stress plays a key role in implant-related inflammation and failure, the ability of nHAp to neutralize free radicals could be advantageous in biomedical applications, particularly in bone tissue engineering and implant coatings.The MTT assay demonstrated that all three nHAp samples were non-cytotoxic to MG63 osteoblast-like cells, with IC₅₀ values above 1000 μg/mL. This suggests excellent biocompatibility, making biologically derived nHAp a viable alternative to synthetic hydroxyapatite. The slight differences in IC₅₀ values among the samples may be due to variations in elemental composition and crystal structure, which can influence their interactions with cells. The hybrid hen-derived nHAp (HE1) exhibited slightly greater bioactivity, potentially due to its mineral content. Compared to the commercial reference sample, the biologically derived nHAp samples exhibited comparable antioxidant activity and minimal cytotoxicity, suggesting that eggshell-derived nHAp could serve as a sustainable alternative to conventionally synthesized hydroxyapatite. Additionally, this method provides an eco-friendly solution for waste management, repurposing poultry industry by-products into valuable biomedical materials. Given these findings, biologically derived nHAp could be explored for use in bone regeneration, dental applications and drug delivery systems. Future research should focus on in vivo biocompatibility studies, mechanical property enhancements and surface modifications to improve osteogenic potential. Moreover, metal doping strategies (e.g., incorporating magnesium or zinc) could further enhance its bioactivity. Overall, this study highlights the potential of biologically derived nHAp as a sustainable and effective biomaterial for biomedical applications. With further optimization and validation through clinical studies, it could serve as a cost-effective alternative for bone implants, coatings and tissue engineering scaffolds.

This study introduces a novel and sustainable approach for synthesizing nanohydroxyapatite (nHAp) from Indian and hybrid hen eggshells, transforming poultry waste into a high-value biomedical material. Unlike conventional chemical synthesis, this eco-friendly method provides a cost-effective and resource-efficient alternative while preserving bioactivity. The antioxidant potential observed in the biologically derived nHAp highlights its ability to mitigate implant-related oxidative stress, a key factor in improving tissue regeneration. Additionally, the non-cytotoxic nature of the synthesized nHAp, confirmed through MTT assays, establishes its biocompatibility and safety for biomedical applications such as bone tissue engineering, implant coatings and dental restorations. This research is distinctive in demonstrating that waste-derived nHAp can match commercial hydroxyapatite in bioactivity, offering a sustainable solution for biomedical engineering. The integration of natural bioactive compounds may further enhance its biological properties, distinguishing it from synthetic alternatives. By bridging the gap between waste management and advanced biomaterials, this study provides a strong foundation for future in vivo research and clinical applications. Its potential impact on regenerative medicine and implantology makes it highly relevant for publication in biomaterials and pharmaceutical sciences journals.

The authors would like to thank Aakaar Biotechnologies Private Limited, Lucknow, Uttar Pradesh, for providing lab facilities and support during research work.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish or preparation of the manuscript.