Biomedical Applications of Zirconia-Based Nanomaterials: Challenges and Future Perspectives

ZrO2 nanoparticles have received substantially increased attention in every field of life owing to their wide range of applications. Zirconium oxide is a commercially economical, non-hazardous, and sustainable metal oxide having diversified potential applications. ZrO2 NPs play a vast role in the domain of medicine and pharmacy such as anticancer, antibacterial, and antioxidant agents and tissue engineering owing to their reliable curative biomedical applications. In this review article, we address all of the medical and biomedical applications of ZrO2 NPs prepared through various approaches in a critical way. ZrO2 is a bio-ceramic substance that has received increased attention in biomimetic scaffolds owing to its high mechanical strength, excellent biocompatibility, and high chemical stability. ZrO2 NPs have demonstrated potential anticancer activity against various cancer cells. ZrO2-based nanomaterials have exhibited potential antibacterial activity against various bacterial strains and have also demonstrated excellent antioxidant activity. The ZrO2 nanocomposite also exhibits highly sensitive biosensing activity toward the sensing of glucose and other biological species.


Introduction
Zirconium is a transition metal with enhanced thermal, mechanical, catalytic, and thermal characteristics and also demonstrates significant corrosion resistance [1]. Zirconium has an atomic number of 40 and has a distinctive physical and chemical properties like titanium [2]. Zirconium exists naturally in five different isotopic forms; out of which 90 Zr exists abundantly in nature (51.45%) [3]. Zirconium dioxide (ZrO 2 ) is also called zirconia and has a monoclinic crystal structure at room temperature [4]. ZrO 2 is an n-type semiconductor and has many fascinating properties such as a high dielectric constant, ion-exchange ability, a high refractive index, high optical transparency, low thermal conductivity, a low coefficient of thermal expansion, polymorphic nature, and exceptional chemical and optical properties [5,6]. ZrO 2 is a material of great technological interest, having good natural color, transformation toughness, good chemical stability, and high strength and being an excellent corrosion-, chemical-, and microbial-resistant material [7]. ZrO 2 is a polymorphic crystal found in three crystallographic forms: monoclinic, cubic, and tetragonal [8]. At room temperature, the monoclinic phase is stable and transforms into the tetragonal phase at 1170 • C, while this phase transforms to the cubic form at 2370 • C [9], which is unstable at ambient temperature in bulk forms [10]. Monoclinic ZrO 2 has a coordination number of seven, while the cubic and tetragonal ZrO 2 have a coordination number of eight. A coordination number of seven is favorable owing to its strong Zr-O covalent bond, and, thus, monoclinic ZrO 2 at lower temperatures is realized to be thermodynamically stable [11]. Considerable efforts have been reported for stabilizing the unstable tetragonal and cubic crystal phases by doping [12]. ZrO 2 is doped with other metal oxides, such as MgO, Y 2 O 3 , CaO, and Ce 2 O 3 , to form stabilized cubic or tetragonal phases [13]. Adding other metal oxides in low amounts as a dopant, the conversion to the monoclinic lattice Zirconium oxide is a commercially economical, non-hazardous, and sustainable metal oxide having diversified potential applications [18]. ZrO2 is considered an important candidate material for advanced ceramics because of its good chemical stability, high strength, and excellent high-temperature performance [19]. ZrO2 holds both reducing and oxidizing properties owing to its basic as well acidic nature and wide bandgap (5.0-5.5 eV) [20,21]. ZrO2 has several advantages over other ceramic materials due to the transformation-toughening mechanism, which provides excellent mechanical properties, such as fracture toughness and fracture strength [22]. Due to these advantages, zirconiabased materials are potentially applied in various advanced fields for photocatalysts [23,24], adsorption [25,26], anti-corrosion coating [27], supercapacitor [28], Li-ion batteries [29], sensors [30,31], water splitting [32], solar cells [33], etc. Research studies on ZrO2based nanoparticles are increasing day by day. Figure 2 presents the number of articles published on ZrO2-based NPs, which has been increasing continuously in 2010-2023. Such an increase in interest in ZrO2-based materials is due to their outstanding properties such as hardness, optical transparency, high refractive index, and chemical and photoelectron stability [34]. ZrO2 ceramic is an advanced biomaterial widely used in medical engineering industries owing to its superior biocompatibility and mechanical strength over other conventional ceramic materials [35]. Zirconium oxide is a commercially economical, non-hazardous, and sustainable metal oxide having diversified potential applications [18]. ZrO 2 is considered an important candidate material for advanced ceramics because of its good chemical stability, high strength, and excellent high-temperature performance [19]. ZrO 2 holds both reducing and oxidizing properties owing to its basic as well acidic nature and wide bandgap (5.0-5.5 eV) [20,21]. ZrO 2 has several advantages over other ceramic materials due to the transformation-toughening mechanism, which provides excellent mechanical properties, such as fracture toughness and fracture strength [22]. Due to these advantages, zirconia-based materials are potentially applied in various advanced fields for photocatalysts [23,24], adsorption [25,26], anti-corrosion coating [27], supercapacitor [28], Li-ion batteries [29], sensors [30,31], water splitting [32], solar cells [33], etc. Research studies on ZrO 2 -based nanoparticles are increasing day by day. Figure 2 presents the number of articles published on ZrO 2 -based NPs, which has been increasing continuously in 2010-2023. Such an increase in interest in ZrO 2 -based materials is due to their outstanding properties such as hardness, optical transparency, high refractive index, and chemical and photoelectron stability [34]. ZrO 2 ceramic is an advanced biomaterial widely used in medical engineering industries owing to its superior biocompatibility and mechanical strength over other conventional ceramic materials [35].

Biomedical Applications of ZrO2 NPs
ZrO2 NPs have been utilized in various applications as antimicrobial agents, nanopowder filling, sintering raw material, nanocoating, and anticancer and antioxidant agents. The functionalization of ZrO2 NPs as hybrid substances has received increased attention in microscale valves, tissue-engineering scaffolds, microfluidic devices, bone prostheses, and drug delivery devices, as well as other medical devices, owing to their bionics and biocompatible and mechanical properties [36]. Bio-medically, ZrO2-based materials are efficiently used in meat packaging [37], dentistry [38], artificial scaffolds [39], etc. At the tissue level, ZrO2 was observed to be biocompatible like titanium. Cultured osteoblasts proliferate and differentiate on zirconia without causing any adverse effects. ZrO2 is a bioinert ceramic material because, after implantation, it shows only a morphological fixation with its surrounding tissues without creating any biological/chemical bonding [40].
ZrO2 is generally synthesized via different chemical approaches such as sol-gel, coprecipitation, solvothermal, hydrothermal, etc. These synthesizing approaches for ZrO2 preparation use toxic chemicals and energy-intensive and costly equipment processes for achieving crystallinity [41]. The green synthesis of ZrO2 NPs excludes the usage of hazardous chemicals, which might generate toxic intermediates in conventional synthesis methods. Plant-mediated nanofabrication is a cost-effective and easy-to-handle approach that does not need any exceptional reaction conditions [18,42]. The biogenic synthesis approach uses affordable and locally available plants and other biocompatible sources, e.g., fungi, algae, and bacteria for the synthesis of ZrO2. The extracted biomolecules from these biological sources act as potent bioreducing, biocapping, and biostabilizing agents and produce ZrO2 in sufficient quantity. This green method for ZrO2 NP synthesis also matches well with the principle of green and sustainable chemistry [43]. In green synthesis, ZrO2 NPs are produced through a reduction process. A mechanism proposed in a study revealed that phytochemical compounds of the -OH moiety present in the plant may carry out the process of reduction. The enol compounds convert into keto form, which releases hydrogen atoms and reduces the ion of the zirconium salt, and form ZrO2 NPs after annealing [44]. Plant extracts are very promising owing to their complex

Biomedical Applications of ZrO 2 NPs
ZrO 2 NPs have been utilized in various applications as antimicrobial agents, nanopowder filling, sintering raw material, nanocoating, and anticancer and antioxidant agents. The functionalization of ZrO 2 NPs as hybrid substances has received increased attention in microscale valves, tissue-engineering scaffolds, microfluidic devices, bone prostheses, and drug delivery devices, as well as other medical devices, owing to their bionics and biocompatible and mechanical properties [36]. Bio-medically, ZrO 2 -based materials are efficiently used in meat packaging [37], dentistry [38], artificial scaffolds [39], etc. At the tissue level, ZrO 2 was observed to be biocompatible like titanium. Cultured osteoblasts proliferate and differentiate on zirconia without causing any adverse effects. ZrO 2 is a bioinert ceramic material because, after implantation, it shows only a morphological fixation with its surrounding tissues without creating any biological/chemical bonding [40]. ZrO 2 is generally synthesized via different chemical approaches such as sol-gel, coprecipitation, solvothermal, hydrothermal, etc. These synthesizing approaches for ZrO 2 preparation use toxic chemicals and energy-intensive and costly equipment processes for achieving crystallinity [41]. The green synthesis of ZrO 2 NPs excludes the usage of hazardous chemicals, which might generate toxic intermediates in conventional synthesis methods. Plant-mediated nanofabrication is a cost-effective and easy-to-handle approach that does not need any exceptional reaction conditions [18,42]. The biogenic synthesis approach uses affordable and locally available plants and other biocompatible sources, e.g., fungi, algae, and bacteria for the synthesis of ZrO 2 . The extracted biomolecules from these biological sources act as potent bioreducing, biocapping, and biostabilizing agents and produce ZrO 2 in sufficient quantity. This green method for ZrO 2 NP synthesis also matches well with the principle of green and sustainable chemistry [43]. In green synthesis, ZrO 2 NPs are produced through a reduction process. A mechanism proposed in a study revealed that phytochemical compounds of the -OH moiety present in the plant may carry out the process of reduction. The enol compounds convert into keto form, which releases hydrogen atoms and reduces the ion of the zirconium salt, and form ZrO 2 NPs after annealing [44]. Plant extracts are very promising owing to their complex chemical composition and their easier extraction. Phytochemicals in plant extract act as reducing, precipitating, and cap-ping agents and thus demonstrate a significant role in controlling the particle shape, size, and phase stability, as well as other characteristics, of NPs [45].

Antioxidant Activity
Antioxidants are several compounds that protect the body from the noxious effect of free radicals, and their protective mechanism is evaluated through scavenging free radicals [46,47]. The antioxidant activity of the ZnO-ZrO 2 heterojunction was determined by scavenging the 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, which show higher activity than the ascorbic acid. The smaller IC 50 values of the ZnO-ZrO 2 heterojunction (149.20 µg mL −1 ) confirm its high antioxidant potential against ABTS free radicals as compared with the standard ascorbic acid (171.04 µg mL −1 ) [48]. Bioinspired ZrO 2 NPs utilizing an aqueous extract of agriculture waste durva grass exhibited excellent antioxidant activity through a DPPH scavenging assay. The ZrO 2 NPs demonstrate higher activity than aqueous durva grass extract and lower activity than standard ascorbic acid. ZrO 2 NPs are e − donors and can convert the free radicals into products that are more stable via a terminating radical chain reaction and also increased radical scavenging inhibition. ZrO 2 NPs displayed 91.2% scavenging inhibition (IC 50 = 130.38 µg/mL) and the aqueous extract showed 54.8% inhibition (IC 50 = 228.61 µg/mL), while the ascorbic acid demonstrated 95% inhibition (IC 50 = 105.78 µg/mL, a little higher than ZrO 2 NPs) [49]. The antioxidant activity of ZrO 2 is size-dependent, and it was reported that nano-ZrO 2 (particle size distribution is 39 nm in water) scavenges about 71.4% of the free radicals using 1 mg and 76.9% at 100 mg, whereas micro-ZrO 2 scavenges about 57.4% and 69.4% of free radicals at 1 mg and 100 mg, respectively [50]. Similarly optimized Fe 3 O 4 -stabilized ZrO 2 NPs exhibited~76% scavenging inhibition capability. The reason for such high antioxidant activity is the efficient transferring of the O atom electron density of ZrO 2 towards the odd electrons, which are positioned at the N atom of the DPPH, while Fe 3 O 4 helps in the effective electron transfer [51]. ZrO 2 NPs prepared using an aqueous extract of Moringa oleifera leaves revealed 69% radical scavenging efficiency determined via DPPH assay [52]. Similarly, C-ZrO 2 , S-ZrO 2, and C-S-ZrO 2 nanocomposites synthesized using the aqueous leaf extract of Plumeria acuminate show an antioxidant activity with an IC 50 value ranging from 177.60 to 359.46 µg/mL as compared to the activity of Gallic acid (standard), which shows an IC 50 of 19.99 µg/mL. The observed antioxidant activity may be credited to the OH groups present in the NPs, which are similar to the phenolic functional group [53]. The collagen protein and calcium carbonate stabilized ZrO 2 NPs show the 96% of radical scavenging activity [54]. Some of the antioxidant activity of ZrO 2 and their composite-based materials are summarized in Table 1.

Antimicrobial Activity
The antimicrobial activity of ZrO 2 NPs, prepared via the sol-gel method, on E. coli and S. aureus bacterial strains were evaluated, and their results showed that the ZrO 2 NPs displayed 25 mm and 27 mm inhibition zones against the E. coli and S. aureus, respectively. The reason for this activity is the increased formation of reactive oxygen species (ROS) that leads to the destruction of bacterial cells [62]. ZrO 2 also exhibits efficient antibacterial activity in polymer nanocomposites, e.g., the PVA-PEG-PVP-ZrO 2 nanocomposites have established efficient antibacterial potential against Gram-positive and Gram-negative bacteria [63]. ZrO 2 NPs displayed a maximum inhibition zone against A. niger (18 mm) and S. aureus (19 mm) using a maximum concentration of 200 µg/mL [64]. Similarly, Zn-ZrO 2 /TiO 2 coatings synthesized on a titanium alloy (Ti6Al4V) surface demonstrate excellent antibacterial activity against S. aureus, as shown in Figure 3. The figure reveals that the other sample-containing plates contain bacterial colonies in large numbers, while the sample Zn-ZrO 2 /TiO 2 coatings almost kill all of the bacterial colonies, indicating their excellent in vitro antibacterial activity. The surface-ionized ions (Zn 2+ or Zr 4+ ) interact with the negatively charged cell membranes, cause the alternation of bacterial cell permeability, and damage the integrity of cell membranes. This will eventually lead to cytosolic leakage and cause bacterial death [65]. Tetragonal and monoclinic ZrO 2 phases were deposited on stainless steel (316 L SS) and effectively protected bacterial invasion against the pathogenic P. aeruginosa and the subsequent biofilm formation [66].  The release of Zr 4+ ions control the spread as well as the growth of bacterial strains. The release of Zr 4+ ions lead to the leaking of the cell wall membrane. The metal ions admitted in the bacterial cell promote the electrostatic interaction and lead to the production of ROS, which deactivate protein and DNA molecules. This damage to protein and DNA in the bacterial system cuts off their communications and food systems and leads to bac- The release of Zr 4+ ions control the spread as well as the growth of bacterial strains. The release of Zr 4+ ions lead to the leaking of the cell wall membrane. The metal ions admitted in the bacterial cell promote the electrostatic interaction and lead to the production of ROS, which deactivate protein and DNA molecules. This damage to protein and DNA in the bacterial system cuts off their communications and food systems and leads to bacterial cell death [67]. The antibacterial effect of ZrO 2 NPs is attributed generally to the rupturing of the outer bacterial membranes by ROS, mostly OH radicals, which results in phospholipid per oxidation and finally causes cell death [68]. The cell wall of the Gram-negative bacteria, e.g., K. pneumonia, is composed of a peptidoglycan thin layer and has a lipid membrane outside. The ROS produced by ZrO 2 NPs are responsible for the microorganism's cell death. The generation of ROS cannot develop any resistance because these species attack different biomolecules and multiple different sites of the microorganism, leading to oxidation and, finally, cell death. An effective result demonstrated by the Gram-negative bacteria might be due to the electrostatic attraction of the positively charged Zr + ions and the more negatively charged cell wall of bacteria, resulting in the rupturing of the cell wall of bacteria and, finally, cell death. Gram-positive bacteria, e.g., S. aureus, have a thick layer of peptidoglycan, which acts as a barrier to the NPs entering the bacterial cell [69]. Table 2 represents the antibacterial activity of ZrO 2 and some of its composite-based nanomaterials.

Anticancer Activity
The promising properties of ZrO 2 NPs such as thermal stability, biocompatibility, and economical production make them superior materials in biological systems. ZrO 2 NPs have been evaluated for their anticancer activity against various cell lines [78]. A ZnO/ZrO 2 /rGO (reduced graphene oxide) nanocomposite prepared via the green ap-proach using ginger rhizome extract displayed effective anticancer activity in humans. The results represent that ZnO/ZrO 2 /rGO NCs display higher anticancer efficacy in lung cancer (A549) cells and human breast cancer (MCF7) and display good cytocompatibility in normal cell lines and breast epithelial (MCF10A) and human lung fibroblasts (IMR90) cells [79]. ZrO 2 /rGO NCs were prepared using the aqueous leaf extract of Andrographis paniculate via a one-pot solvothermal green synthetic approach, showed excellent anticancer activity toward human A549 and HCT116 cancer cell lines, and did not cause any adverse effect on normal cells (hMSCs) [80]. ZrO 2 NPs synthesized using Eucalyptus globulus leaf extract as an efficient reducing as well as capping agent demonstrate efficient anticancer activities towards the tested cell lines, e.g., A-549 lung cancer cell lines and HCT-116 colon cancer [81].

Bone Tissue Engineering
ZrO 2 is a kind of bio-ceramic material that has received increased attention in biomimetic scaffolds owing to its great chemical stability, excellent biocompatibility, and high mechanical strength. ZrO 2 has been used widely in the field of bone tissue engineering owing to its excellent properties, such as in film or coating on other implants, bone cement, bone graft substitutes, dental prosthesis, and implants, and is therefore preferred as a significant bio-ceramic material in bone repair. Zirconia-based nanocomposites are used widely in bone tissue materials owing to their wear resistance, high mechanical strength, and lowtemperature sintering properties [82]. Zirconium oxide is classified as a bioinert ceramic because it is only morphologically fixed with the surrounding tissue after implantation and does not provide chemical or biological bonding [83]. Porous ZrO 2 scaffolds can be utilized for the restoration of large bone defects because of their favorable biocompatibility, chemical bioinertness, and mechanical strength. Owing to the non-degradable properties of ZrO 2 , its scaffold can assist as a permanent implant material, which provides suitable mechanical support for the tissues as well as a host environment for cell infiltration, waste disposal, nutrient transport, new tissue generation, etc. [84].
Y-ZrO 2 (Yttria-stabilized zirconia) is a stable material having superior mechanical properties, biocompatibility, and an anti-corrosive nature, suggesting its efficient suitability as the in vivo best choice for bone regeneration-based applications over an extended duration [85]. BCP (biphasic calcium phosphate) scaffold reinforced with ZrO 2 was fabricated via fused deposition modeling for bone tissue engineering. BCP scaffold containing 10 wt% ZrO 2 powder had higher compressive strength. The BCP/ZrO 2 scaffold exhibited efficient biocompatibility on MG63 cell proliferation for 7 days. Human mesenchymal stem cells exhibited great viability on the BCP/ZrO 2 scaffolds over 21 days in culture [86]. The ZrO 2 /β-TCP scaffold porosity was adjusted from 65% to 84%, while the compressive strength increased to 6.25 MPa from 4.95, when the ZrO 2 amount was increased from 30 to 50 wt%. The in vitro study revealed that an osteoblasts-loaded ZrO 2 /β-TCP scaffold provided a suitable 3D environment for osteoblast survival and enhanced bone regeneration. The SEM images of the differentiated cells cultured in the scaffolds having different compositions are demonstrated in Figure 4. The osteocyte cell adhesion as well as proliferation in the ZrO 2 /b-TCP scaffold was significantly improved in the ZrO 2 .Y2O3/b-TCP: 30/70 samples [87]. Similarly, a porous magnetic-zirconia calcium bio-nanocomposite scaffold placed in the simulated body fluid displayed the formation of a bone-resembling apatite layer on the surfaces of the nanocomposite having a higher content of magnetic NPs (Fe 3 O 4 ). A biocompatibility assessment revealed that composite scaffolds did not display any toxicity in contact with bone marrow stem cells and increased the growth and proliferation of cells [88]. The other reported ZrO 2 -based materials used in bone tissue engineering are ZrO 2 -SiO 2 ceramic composites [89], TiO 2 -ZrO 2 nanocomposites [90], ZrO 2 /RGO and ZrO 2 /RGO/HA [39], zirconia/hydroxyapatite ceramic composites [91], MWCNTs/ZrO 2 -CaO/Poly(methyl methacrylate) biocomposite [92], ZrO 2 -nanoparticle-doped CTS-PVA-HAP composites [93], Hap-ZrO 2 -Hbn biocomposites [94], Zirconia-toughened hydroxyapatite biocomposites [95], etc.

Dentistry
ZrO2 has been introduced in dentistry due to its superior biomechanical properties (strength, toughness, fatigue resistance, low elasticity module, and fracture strength), biocompatibility, excellent wear resistance, and its similar color to natural tooth [96,97]. Due to the morphological fixation with their surrounding tissues without forming any biological or chemical bonding, ZrO2 has been investigated for dentistry applications [98]. As the demand for cosmetic dental procedures has increased, ZrO2 has become a popular material due to its better biocompatibility, pleasing appearance, strong oxidation resistance, and improved mechanical properties. Moreover, ZrO2 has not been associated with any allergic reactions. Technological advancements in artificial intelligence and machine learning have enabled the development of innovative biological applications of ZrO2 in dental devices. The increasing interest in applying AI in ZrO2 research and therapy is due to its capability to analyze data and identify correlations between seemingly unrelated events [99]. The zirconia types, which are recently introduced in the market, are

Dentistry
ZrO 2 has been introduced in dentistry due to its superior biomechanical properties (strength, toughness, fatigue resistance, low elasticity module, and fracture strength), biocompatibility, excellent wear resistance, and its similar color to natural tooth [96,97]. Due to the morphological fixation with their surrounding tissues without forming any biological or chemical bonding, ZrO 2 has been investigated for dentistry applications [98]. As the demand for cosmetic dental procedures has increased, ZrO 2 has become a popular material due to its better biocompatibility, pleasing appearance, strong oxidation resistance, and improved mechanical properties. Moreover, ZrO 2 has not been associated with any allergic reactions. Technological advancements in artificial intelligence and machine learning have enabled the development of innovative biological applications of ZrO 2 in dental devices. The increasing interest in applying AI in ZrO 2 research and therapy is due to its capability to analyze data and identify correlations between seemingly unrelated events [99]. The zirconia types, which are recently introduced in the market, are effectively commercialized for dental rehabilitation such as inlay, crowns, veneers, onlay (VINCRON), and also in fixed partial dentures [100].
The addition of ZrO 2 to a 3D-printed resin significantly improved the antimicrobial capability of the resulting resin without causing any cellular side effects. This modification has an auspicious future in the field of dentistry for fabricating long-term provisional restorations [101]. The Ti-Zr alloy has high corrosion resistance due to the formation of ZrO 2 and TiO 2 on the surface and their combined effect [102]. A study revealed that introducing 5% glass fillers and 10-20% ZrO 2 NPs efficiently improves the biocompatibility and flexural strength of the dental resin material. The addition of 10%, 20% ZrO 2 , and 5% glass silica by weight meaningfully increases the flexural strength of the resulting 3D-printed resins [103]. ZrO 2 NP coatings to teeth also strengthen the teeth externally and increase their lifespan. ZrO 2 NPs would bind on the surface of bacteria and stop their metabolic activity with food, thus preventing acid synthesis and enamel corrosion because acid penetrates teeth, dissolves enamel, and causes cavity formation. This protection phenomenon can be understood from Figure 5 [104]. Various nanocomposites of ZrO 2 are reported for their utilization in dental applications such as PMMA-ZrO 2 nanocomposites [105], 3D-printed resin reinforced with modified ZrO 2 NPs [106], etc.
The addition of ZrO2 to a 3D-printed resin significantly improved the antimicrobial capability of the resulting resin without causing any cellular side effects. This modification has an auspicious future in the field of dentistry for fabricating long-term provisional restorations [101]. The Ti-Zr alloy has high corrosion resistance due to the formation of ZrO2 and TiO2 on the surface and their combined effect [102]. A study revealed that introducing 5% glass fillers and 10-20% ZrO2 NPs efficiently improves the biocompatibility and flexural strength of the dental resin material. The addition of 10%, 20% ZrO2, and 5% glass silica by weight meaningfully increases the flexural strength of the resulting 3D-printed resins [103]. ZrO2 NP coatings to teeth also strengthen the teeth externally and increase their lifespan. ZrO2 NPs would bind on the surface of bacteria and stop their metabolic activity with food, thus preventing acid synthesis and enamel corrosion because acid penetrates teeth, dissolves enamel, and causes cavity formation. This protection phenomenon can be understood from Figure 5 [104]. Various nanocomposites of ZrO2 are reported for their utilization in dental applications such as PMMA-ZrO2 nanocomposites [105], 3Dprinted resin reinforced with modified ZrO2 NPs [106], etc.

Biosensing
Zirconia is an attractive material due to its high bioactivity for biomolecules [107]. ZrO2 exhibits high stability under surrounding conditions such as pH, temperature, and moisture and demonstrates potential biosensing application [108]. The properties of ZrO2 such as low toxicity, high chemical inertness, environment friendly nature, thermal stability, cost-effective production, biocompatibility, and electrochemical activity pave its way

Biosensing
Zirconia is an attractive material due to its high bioactivity for biomolecules [107]. ZrO 2 exhibits high stability under surrounding conditions such as pH, temperature, and moisture and demonstrates potential biosensing application [108]. The properties of ZrO 2 such as low toxicity, high chemical inertness, environment friendly nature, thermal stability, cost-effective production, biocompatibility, and electrochemical activity pave its way to being a superior electrode material in the sensing of various substances [109]. ZrO 2 NPs have received increased attention in bio-analytical applications due to their lack of toxicity, chemical inertness, and affinity for oxygen-containing groups [110] and have been used In modified electrodes [111]. Along with these distinguished properties, ZrO 2 NPs have been investigated for the development of biosensors [112]. However, some limitations associated with ZrO 2 NPs such as the high aggregation tendency, lower conductivity, and absence of desired functional groups reduce its biosensing and electrochemical performance. Therefore, hybrid systems are highly recommended, which can improve the biosensing and electrochemical characteristics by utilizing the full potential of ZrO 2 NPs [113]. Zr-based coordination polymers have high stability and can be endowed with activity for electrochemical sensing [114]. A mesoporous ZrO 2 -Ag-G-SiO 2 and In 2 O 3 -G-SiO 2 (G for graphene oxide) biosensor were found to be highly selective in detecting E. coli bacteria and could identify an individual E. coli cell in 1 µL volume of the sample within 30 s [115]. CeO 2 -ZrO 2 hollow nanospheres and chitosan composite film were deposited on a Au electrode and used for fabricating a DNA biosensor. The study suggested that the targeted DNA could be detected over a wide range of 1.63 × 10 −13 M to 1.63 × 10 −8 M with a 1.0 × 10 −13 M detection limit using methylene blue dye as an electrochemical indicator. The elaborated fabrication of the composite and the detection mechanism for the designed DNA biosensor are illustrated in Figure 6 [116]. Various ZrO 2 -based materials are utilized for biosensing applications, such as ZrO 2 /Chitosan composites [117,118], nZrO 2 @PC [119], CeO 2 -ZrO 2 composites [120], ZrO 2 @CuNCs [121], Gox-PLL/RGO-ZrO 2 composites [122], TiO 2 -ZrO 2 nanocomposites [123], GQDs@La 3+ @ZrO 2 [124], ChOx/Cu 2 O@MnO 2 -ZrO 2 @AuNPs/GCE [125], ZrO 2 NPs in 1-butyl-3-methylimidazolium trifluoroacetate [126], ZrO 2 -NPs/MacroPSi EGFET [127], polyaniline-graphene oxide composites decorated with ZrO 2 NPs [128], etc.
to being a superior electrode material in the sensing of various substances [109]. ZrO2 NPs have received increased attention in bio-analytical applications due to their lack of toxicity, chemical inertness, and affinity for oxygen-containing groups [110] and have been used In modified electrodes [111]. Along with these distinguished properties, ZrO2 NPs have been investigated for the development of biosensors [112]. However, some limitations associated with ZrO2 NPs such as the high aggregation tendency, lower conductivity, and absence of desired functional groups reduce its biosensing and electrochemical performance. Therefore, hybrid systems are highly recommended, which can improve the biosensing and electrochemical characteristics by utilizing the full potential of ZrO2 NPs [113]. Zr-based coordination polymers have high stability and can be endowed with activity for electrochemical sensing [114]. A mesoporous ZrO2-Ag-G-SiO2 and In2O3-G-SiO2 (G for graphene oxide) biosensor were found to be highly selective in detecting E. coli bacteria and could identify an individual E. coli cell in 1 µL volume of the sample within 30 s [115]. CeO2-ZrO2 hollow nanospheres and chitosan composite film were deposited on a Au electrode and used for fabricating a DNA biosensor. The study suggested that the targeted DNA could be detected over a wide range of 1.63 × 10 −13 M to 1.63 × 10 −8 M with a 1.0 × 10 −13 M detection limit using methylene blue dye as an electrochemical indicator. The elaborated fabrication of the composite and the detection mechanism for the designed DNA biosensor are illustrated in Figure 6 [116]. Various ZrO2-based materials are utilized for biosensing applications, such as ZrO2/Chitosan composites [117,118], nZrO2@PC [119], CeO2-ZrO2 composites [120], ZrO2@CuNCs [121], Gox-PLL/RGO-ZrO2 composites [122], TiO2-ZrO2 nanocomposites [123], GQDs@La 3+ @ZrO2 [124], ChOx/Cu2O@MnO2-ZrO2@AuNPs/GCE [125], ZrO2 NPs in 1-butyl-3-methylimidazolium trifluoroacetate [126], ZrO2-NPs/MacroPSi EGFET [127], polyaniline-graphene oxide composites decorated with ZrO2 NPs [128], etc.

Challenges
Due to polymorphism, pure ZrO2 cannot be utilized in any application owing to its volume expansion (4-5%) occurring during cooling. This can be prevented by stabilizing oxides such as MgO, CaO, Sc2O3 CeO2, and Y2O3, which are used for retaining their metastable tetragonal phase [129]. Doping of a noble metal and/or metal oxide into ZrO2 is also a feasible approach for minimizing the drawbacks associated with ZrO2 [130]. Due to the extensive use of NPs, they can enter the environment via many routes, undergo

Challenges
Due to polymorphism, pure ZrO 2 cannot be utilized in any application owing to its volume expansion (4-5%) occurring during cooling. This can be prevented by stabilizing oxides such as MgO, CaO, Sc 2 O 3 CeO 2, and Y 2 O 3, which are used for retaining their metastable tetragonal phase [129]. Doping of a noble metal and/or metal oxide into ZrO 2 is also a feasible approach for minimizing the drawbacks associated with ZrO 2 [130]. Due to the extensive use of NPs, they can enter the environment via many routes, undergo transformations, and pose toxicity to organisms in different environmental compartments [131].
NPs may cross different cellular barriers when entering the human body and ultimately reach the most sensitive organs such as the lung, liver, and kidney. This phenomenon may result in DNA mutations, mitochondrial damage, and, eventually, cell death [132].

Future Perspectives
The extensive use of ZrO 2 NPs suggests the dire need to evaluate their adverse effects on the biological systems because limited literature is reported on the evaluation of toxic behaviors of ZrO 2 -based nanomaterials (NMs) with respect to cytotoxicity, bioactivity, and antioxidant activity [50]. Environmental issues should be considered before using ZrO 2 NMs for any biomedical applications, which can cause environmental hazards and can also affect livings things. It is highly suggested to carry out theoretical simulations along with performing experimental work because DFT calculations help to predict in advance the desired goal and suggested mechanism and support the experimental results. It is suggested to evaluate different biological activities and applications of ZrO 2 NMs, which will lead to their multifunctional behaviors that will increase their medical value. In biosensing applications, the selectivity of ZrO 2 -based biosensors is very important for their accurate and precise sensing. Manipulation of the size, shape, and morphology of the ZrO 2 NPs could lead to achieving optimized activities in various biological applications because these parameters greatly affect their activity. Controlling these parameters can achieve the desired goals in biological applications.

Conclusions
Due to its non-hazardous nature, excellent properties, and diversified applications, ZrO 2 is considered an important candidate material in the fields of medicine and pharmacy. ZrO 2 NP-based nanomaterials with extraordinary characteristics have been used in the recent area of biomedicine, owing to their non-toxic nature, excellent biocompatibility, and high chemical stability. ZrO 2 NP-based NMs exhibited outstanding antibacterial, anticancer, and antioxidant activities due to their unique biological properties. The precise biosensing applications of ZrO 2 towards glucose and other biological species are due to its high bioactivity for biomolecules and high stability under surrounding conditions such as pH, temperature, moisture, etc. In the future, assessing the toxicity of ZrO 2 NPs before evaluating their biological applications is highly recommended.

Conflicts of Interest:
The author declares no conflict of interest.