Carbon Dot Nanozymes in Orthopedic Disease Treatment: Comprehensive Overview, Perspectives and Challenges

Abstract

Nanozymes, as a new generation of artificial enzymes, have attracted increasing attention in the field of biomedicine due to their multiple enzymatic characteristics, multi-functionality, low cost, and high stability. Among them, carbon dot nanozymes (CDzymes) possess excellent enzymatic-like catalytic activity and biocompatibility and have been developed for various diagnostic and therapeutic studies of diseases. Here, we briefly review the representative research on CDzymes in recent years, including their synthesis, modification, and applications, especially in orthopedic diseases, including osteoarthritis, osteoporosis, osteomyelitis, intervertebral disc degenerative diseases, bone tumors, and bone injury repair and periodontitis. Additionally, we briefly discuss the potential future applications and opportunities and challenges of CDzymes. We hope this review can provide some reference opinions for CDzymes and offer insights for promoting their application strategies in the treatment of orthopedic disease.

1. Introduction

Nanozymes were proposed by Yan’s group in 2007, which was the first to demonstrate that ferromagnetic nanoparticles (Fe₃O₄ NPs) possess peroxidase (POD)-like activity [1]. With the continuous advancement and improvement in nanotechnology, an increasing number of nanomaterials, such as carbon dots (CDs) [2], metal oxides [3,4], and metal-organic frameworks [5,6], have been reported to possess various enzyme-like catalytic properties. Recently, increasing attention has been given to nanozymes because of their multienzyme-like activity, multifunctionality, high stability, and low cost [7,8]. They can regulate substances such as reactive oxygen species (ROS) and reactive nitrogen species, which play important roles in life activities and thereby play important roles in the treatment of various diseases. Among them, carbon dot nanozymes (CDzymes) are emerging as promising nanomaterials. The catalytic performance of carbon dots stems from the formation of internal cavities and the transfer of electrons. By adjusting the surface groups or the molecular structure of the carbon core, carbon dots can acquire specific catalytic capabilities [9]. Owing to their simple synthesis method, environmental friendliness, and low cost, CDzymes are widely used to treat various diseases, including local inflammation [10], infectious diseases [11], neurodegenerative diseases [12,13], dental diseases [14], and tumors [15]. This article reviews the research progress of CDzymes in orthopedic diseases. The diseases covered in this review include osteoarthritis, osteoporosis, osteomyelitis, intervertebral disc degenerative diseases, bone tumors, bone defects, periodontitis, etc. (Figure 1). CDzymes offer the following advantages for the treatment of these diseases. First, in conditions such as osteoarthritis or intervertebral disc degeneration, the affected areas are located in avascular or hypovascular closed environments. Owing to their extremely small size, CDzymes can diffuse more effectively in these affected areas. Second, owing to the lack of blood vessels, CDzymes can remain in affected areas for longer periods, thereby increasing their efficacy. Third, compared with currently used clinical drugs, CDzymes exhibit a superior biocompatibility and lower organ toxicity. Finally, the potential applications and main challenges of CDzymes are discussed. This review not only provides some guiding perspectives for the development of CDzymes but also promotes the development of enzyme-mimicking therapeutic strategies for orthopedic diseases.

2. Preparation Methods for CDzymes

2.1. Top-Down Approach

The top-down approach aims to physically separate or break down large carbon structures into quantum-level CDzymes with a high crystallinity and regular surfaces [16]. These synthesis strategies mainly include laser etching, arc discharge, oxidative decomposition, hydrothermal methods, solvothermal methods, and electrochemical methods. Qu et al. used multiwalled carbon nanotubes as the carbon source and employed the reflux oxidation method with concentrated nitric acid to prepare oxidized graphene quantum dots. The size distribution of these CDzymes was approximately 3.2 nm, and they exhibited excellent peroxidase activity over a wide pH range and were applied for efficient glucose detection [17]. They discovered that the carboxyl and carbonyl groups on the CDzymes are the binding sites and active sites for the substrates during the catalytic process and that the hydroxyl groups inhibit the enzyme-like activity. Therefore, CDzymes with a relatively high degree of surface oxidation have a relatively high catalytic activity, similar to enzymes. Although the oxidative cracking method can efficiently produce CDzymes, its reaction environment is rather demanding, requiring a large amount of acids, bases, and oxidants. Therefore, green synthesis becomes extremely important. For example, Jiang et al., inspired by the anti-inflammatory properties of traditional Chinese medicines, synthesized CDzymes (SB-HHD-CDs). The SB-HHD-CDs exhibit a remarkable pH and thermal stability and can rapidly eliminate various nitrogen species (RONs) and reactive oxygen, such as superoxide anions, hydroxyl radicals, 1,1-diphenyl-2-pyridinone radicals, and nitric oxide radicals. The prepared CDs were used as an economical and effective additive and integrated into a cigarette filter to effectively remove over 80% of the RONS produced by burning cigarettes [18]. Zeng et al. reported a biocompatible nanozyme, which was prepared from a bioactive natural product in coffee, chlorogenic acid (ChA). The green quinone CDs exhibited significant glutathione peroxidase-like activity and promoted ferroptosis in cancer cells by interfering with the lipid repair system catalyzed by GPX4. The green quinone CDs significantly inhibited tumor growth in HepG2 tumor-bearing mice, with almost no side effects. In particular, in H22 tumor-bearing mice with liver cancer, green quinone carbon dots recruited large numbers of tumor-infiltrating immune cells, including T cells, macrophages, and NK cells, thereby transforming “cold” tumors into “hot” tumors and activating the systemic antitumor immune response [15].

2.2. Bottom-Up Approach

The bottom-up approach refers to the polymerization of small molecules or low-molecular-weight polymer precursors composed of functional groups such as carboxyl, hydroxyl, and amino groups, followed by carbonization [11,19]. This method mainly includes pyrolysis, microwave heating, hydrothermal/solvothermal methods, etc. By adopting a bottom-up approach, the physicochemical properties of CDzymes can be regulated through the selection of precursors, thereby influencing their enzyme-mimicking activities. Among them, hydrothermal methods and pyrolysis methods are the two most commonly used approaches. Hydrothermal synthesis is a chemical reaction carried out in aqueous solution, typically at temperatures above 100 °C and under high pressure. Pyrolysis is the process of decomposing organic matter in an inert atmosphere at high temperatures and controlled pressure. Among them, the hydrothermal method has the lowest cost and is simple and efficient. When CDzymes are synthesized at lower temperatures (below 250 °C) through a bottom-up approach, the molecular structure of the precursor is not completely destroyed; thus, many functional groups are retained. Studies have shown that CDzymes synthesized from the same precursor via the bottom-up approach do not significantly differ in size or morphology, but their enzyme-like activities and reaction yields significantly differ. Huang et al. prepared metal-free CDzymes with peroxidase (POD)-like, superoxide dismutase (SOD)-like, and catalase (CAT)-like activities through a hydrothermal method in which p-phenylenediamine and ethylenediamine were used as precursors. CDzymes were injected into mice with liver inflammation and effectively reduced ROS production and proinflammatory cytokines [20]. Compared with the pyrolysis method for preparing CDzymes, the hydrothermal method is more convenient, environmentally friendly, and green. Xu et al. developed a simple and highly efficient strategy to prepare selenium-doped carbon quantum dots (Se-CQDs) by conducting the hydrothermal treatment of selenocysteine. The selenium heteroatoms endow Se-CQDs with reversible redox-dependent fluorescence properties. Moreover, the free radical •OH can be effectively eliminated by Se-CQDs. Highly harmful ROS within cells are reduced after Se-CQDs are internalized into cells. This characteristic enables Se-CQDs to protect biological systems from the effects of oxidative stress [21]. The advantages and disadvantages of the different methods are provided in

Table 1. The advantages and disadvantages of different methods.

	Synthesis Methods	Advantages	Disadvantages	Ref.
Top-Down	In situ pyrulysis	High yield, cheap raw materials	Ultra-high temperature, easy to aggregate	[16]
	Oxidation reflux	High purity and high yield	Strong acid and strong alkali environment	[17]
Bottom-Up	Hydrothermal	Easy control of both size and shape, high purity	High pressure and high heating temperature	[21]
	Microwave	Reaction conditions environmentally friendly	Low yield	[22]

. Each method has its own advantages, and the choice mainly depends on the selection of raw materials. In general, the bottom-up strategy offers a wide range of available raw materials and makes it relatively easy to achieve large-scale production.

3. Performance Regulation of CDzymes

Like natural enzymes, the catalytic performance of CDzymes is also influenced by numerous factors, including mainly the physical and chemical properties of the CDzymes themselves (such as surface functional groups, microstructure, and composition, etc.) and the external environment (such as temperature, pH, and light, etc.). Next, we systematically summarize and discuss various strategies for regulating the enzyme-like activities of CDzymes.

3.1. Element Doping

Doping is an effective method to improve the catalytic performance of CDzymes [23,24]. Introducing specific element atoms or ions into the main carbon nanomaterials can regulate the electronic structure and geometric structure of the nanomaterials, promoting the transport of substances and the transfer of charges, thereby enhancing their enzyme-like catalytic performance. Doping with nonmetallic elements (such as N and S) is an efficient nonmetallic doping strategy. By taking advantage of the electronegativity differences of the dopants, the electronic structure of the central atoms can be adjusted, leading to changes in the energy barriers of active intermediates, thereby improving the catalytic performance of CDzymes [25,26]. Kang et al. synthesized nitrogen-doped carbon dots (TA-NCDs) via a hydrothermal method, as shown in Figure 2a. Their catalytic activity is similar to that of an oxidase and can be triggered by visible light (420 nm to 700 nm). As a CDzyme, the maximum reaction rate (Vmax) of TA-NCDs is 121.95 × 10⁻⁸ moles per s, and the Michaelis-Menten constant (km) is 0.61 millimoles. In situ transient photocurrent testing confirmed that pyrrole nitrogen is the catalytic active site and the substrate binding site in TA-NCDs. Upon visible light exposure, the oxidase-like activity of TA-NCDs significantly increased the level of ROS in the cells, thereby enabling them to exhibit biological activity under visible light induction and to kill up to 60% of HeLa cells [27]. Chen et al. prepared positively charged carbon dots (CDs) with p-phenylenediamine and polyethyleneimine as raw materials via a simple one-pot solvothermal method, as shown in Figure 2b. The N-doped CDs exhibited excellent biocompatibility in in vitro cell toxicity tests, hemolysis tests, and in vivo toxicity assessment. The positively charged CDs showed remarkable antibacterial effects on S. aureus at extremely low concentrations, reducing the risk of wound infection. Moreover, surface defects and unpaired electrons endow CDs with an excellent performance, such as effectively eliminating excessive free radicals, alleviating oxidative stress damage, accelerating the transition from the inflammatory–proliferative phase of wounds, and promoting wound healing. Skin infection in a mouse model demonstrated that CDs could effectively promote the healing of infected skin wounds by simply dripping or spraying them onto the wounds without obvious side effects [28]. When multiple heteroatoms are codoped, the cooperative effect among the heteroatoms also has a significant effect on the catalytic performance of the CDzyme. Saha et al. successfully synthesized nitrogen- and sulfur-doped carbon dots (N, S-CDs) via a solvent-free and microwave-assisted method, as shown in Figure 2c. Their studies have shown that the S and N doping of carbon dots (with an energy band gap of 2.11 electron volts) enables 3,3,5,5′-tetramethylbenzidine (TMB) to undergo photooxidation reactions under extended visible light excitation at a pH value of 4. The photo-oxidase activity generated by S- and N-doped carbon dots produced a Michaelis constant (km) of 1.18 millimoles and a maximum initial velocity (Vmax) of 4.66 × 10⁸ mL/s under 525 nm illumination. Moreover, visible light irradiation can also induce bactericidal effects and suppress the growth of Escherichia coli (E. coli) [22].

In addition to nonmetallic elements, transition metal atoms (such as zinc, iron, copper, cobalt, and manganese) or their ions can be doped into CDzymes to directly form catalytic active sites, thereby enhancing their catalytic performance [29,30,31]. Wang et al. synthesized novel magnetofluorescent Mn-CDs. They exhibit an excellent physiological stability, near-infrared (NIR) luminescence properties, a high relaxation rate, and efficient ¹O₂ generation capability. More importantly, the Mn-CDs can efficiently catalyze the generation of oxygen from H₂O₂ and successfully improve the hypoxic conditions of tumors, thereby increasing the efficiency of photodynamic therapy (PDT). This magnetic fluorescent Mn-CD can be used as an acid H₂O₂-driven oxygen generator for dual-modal fluorescence/magnetic resonance imaging, and it effectively enhances the photodynamic treatment effect on hypoxic solid tumors through in situ oxygen generation [32]. In addition to the amount of metal doping, the type of metal doping also affects the catalytic activity of the CDzymes [33,34]. Zhan et al. prepared single-atom carbon dots with a CuO₄ coordination environment (Cu-SLCDs) and a CuN₄ coordination environment (CuN-CDs). Both of these materials exhibited similar multienzyme (oxidase, peroxidase, superoxide dismutase, and catalase) activities, among which the Cu-SLCDs presented relatively high activity levels. Compared with the CuN-CDs, the polyphenol p-π conjugated structure created a unique electron donation coordination environment, thereby increasing the electronic density at the Fermi level of the Cu-SLCDs, enhancing electron transfer, and improving the multienzyme-like activity. Cu-SLCDs effectively eliminated more than 98.6% of bacteria at a concentration of 25 µg mL⁻¹, while reducing the levels of ROS in normal cells and promoting effective wound healing [35]. In addition, Lin et al. introduced single-atom iron into carbon dots (SA Fe-CDs) through an in situ pyrolysis method. The dispersed iron centers of the atoms maximize the utilization efficiency and effectively ensure the high OXD-like activity of the Fe-CDs, which can catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to the blue oxidation product (TMBox). Owing to the internal filtering effect, the generated TMBox can inhibit the inherent fluorescence of the Fe-CDs. If Pi is present, it can inhibit the OXD-like activity of Fe-CDs through coordination with iron mononuclear sites, thereby inhibiting the catalytic TMB colorimetric reaction and the recovery of the fluorescence signal. Through this strategy, the chromatic detection and fluorescence detection of Pi are achieved, with excellent performance [16]. Furthermore, introducing other transition metals into single-metal CDzymes is also an effective method of regulating the catalytic performance of CDzymes. The reasonable incorporation of metals can alter the electronic properties of the host and guest molecules and generate new catalytic active sites, effectively enhancing catalytic activity through synergistic effects. Zhou et al. proposed a multifunctional nano-enhancer Bi, Mn-CD-FA@CaP, which achieves a deep penetration and high tumor accumulation ability through dynamic conformational changes, as shown in Figure 3a. Bi, Mn-CD-FA@CaP is a bismuth–manganese-doped CD material that possesses radiosensitizing properties and a similar catalytic activity to catalase and is capable of generating O₂. Folic acid (FA) modification enhances the ability of a material to target tumor cells. Calcium phosphate nanoparticles encapsulate the CDs to form Bi, Mn-CD-FA@CaP. Through the enhanced permeability and retention (EPR) effect, improved performance is shown in in vivo tumor accumulation. When encountering an acidic tumor microenvironment, the encapsulated structure degrades, releasing smaller Bi,Mn-CD-FA particles, which can deeply penetrate the tumor tissue. Their experiments confirmed that Bi, Mn-CD-FA@CaP can effectively target tumor tissues, enhance radiotherapy-induced apoptosis, and inhibit tumor growth. Notably, the nanoparticles achieve a 40% tumor cure rate and ensure a 100% survival rate within 45 days after treatment [36]. On the other hand, owing to the clear active sites of natural enzymes, mimicking the characteristics of the active sites of natural enzymes to prepare CDzymes is also an efficient approach. Wu et al. reported biocompatible -Cu-O-Zn bimetallic covalently doped carbon dots (CuZn-CDs), as shown in Figure 3b. They possess the activities of CAT and SOD, mainly because of their abundant electrons and electron transfer ability. Moreover, the doped copper helps to rebalance the positive charge of the zinc dopant caused by low pH values, enabling the CuZn-CDs to exhibit catalase activity rather than superoxide dismutase activity. Therefore, the CuZn-CDs demonstrated sufficient ROS-scavenging ability in vitro and protected cardiomyocytes from ROS-induced damage. The in vivo results further indicated that the heart could be protected from ischemia-reperfusion injury by the CuZn-CDs. In addition to antioxidant treatment, the low toxicity and rapid renal clearance characteristics of the CuZn-CDs are also reflected in the following regard: animal experimental models have shown that they have an extremely high biocompatibility, which is beneficial for their application in clinical practice [37].

3.2. Surface Modification

An increasing number of studies have shown that the types, quantities, and charges of the surface groups of CDzymes significantly influence their physical and catalytic properties. Therefore, the catalytic performance of CDzymes can be adjusted through surface modification. Goswami et al. used various carbon-containing molecules with different functional groups, such as L-glutamic acid, glycine, and succinic acid, as model compounds. Through theoretical simulation and experimental determination, they reported that hydroxyl groups are unfavorable for enzyme-like catalysis, whereas carboxyl groups can serve as binding sites and catalytic sites for enzyme-like substrates. Among these model compounds, L-glutamic acid has not only many carboxyl groups but also an α-amino group that stabilizes the carboxyl group, thus resulting in a relatively high enzyme-like POD catalytic activity. They prepared photoluminescent carbon dots (CDs) with a pseudo-POD activity using L-glutamic acid as the raw material. The charges on the surface of the CDs could be adsorbed by ABTS through electrostatic interactions. After H₂O₂ was added, ABTS could be rapidly oxidized, and the absorbance of the system increased, thereby achieving the colorimetric detection of H₂O₂. In addition, the oxidized ABTS could be released from the surface of the CDs due to the change in charge, so the CDs could be recycled into the next catalytic oxidation process. Surface modification can also improve the catalytic activity by adjusting the affinity between the nanozyme and the substrate [38]. Shen et al. reported that for TMB, citric acid (CA)-modified carbon dots (CA-CDs) have a greater affinity than polyethyleneimine (PEI)-modified carbon dots (PEI-CDs). Therefore, when TMB is used as the substrate, CA-CDs exhibit increased mimetic POD activity. Conversely, for ABTS, PEI-CDs have a higher affinity than CA-CDs do; thus, PEI-CDs exhibit a higher mimetic POD activity when ABTS is used as the substrate. Moreover, both CA-CDs and PEI-CDs exhibit a higher catalytic activity than unmodified CDs do [39]. Owing to the different interactions between different groups and the substrate, the catalytic activity of CDzymes can be improved by increasing the quantity of specific functional groups. Wang et al. constructed GSH-CDs (glutathione-modified carbon dots), which are a new type of carbon dot antioxidant CDzyme. They are used to remove a large amount of ROS to maintain the stability of the nuclear tissue at the physical redox level. By exerting antioxidant effects (such as SOD, CAT, glutathione peroxidase (GPx), and total antioxidant capacity), GSH-CDs, which have a good biocompatibility, can significantly eliminate endogenous reactive oxygen and have been proven to effectively rescue mitochondrial dysfunction and protect nuclear cells from aging, catabolism, and inflammatory factors. In vivo imaging results and histological indicators (such as the intervertebral disc height index (DHI) and Pfirrmann score) indicated that after the local application of the GSH-CDs, the progression of IVDD significantly improved. GSH-CDs have excellent potential to prevent mitochondrial damage to neural precursor cells caused by an excessive accumulation of reactive oxygen and promote the progression of IDD, providing a potential treatment option for clinical therapy [40].

3.3. Combined with Other Materials

CDzymes with a single catalytic function often fail to meet the demands of various fields. If they are combined with other nanomaterials with special properties to form a composite material and these synergistic or enhancing effects are utilized to increase the catalytic activity of the CDzymes, the characteristics of each component can be leveraged to enrich the functions of the composite material. This can broaden the application of CDzymes in catalysis and biomedical fields, etc. For example, Wang et al. combined CDzymes with a POD-like activity and lactate oxidase activity. First, the large amount of lactate accumulated at the tumor site was converted into H₂O₂ substrate by lactate oxidase. Through the peroxidase reaction between CDzymes and H₂O₂, highly toxic hydroxyl radicals were subsequently generated for efficient tumor treatment research [41]. Shuai et al. synthesized Mn-CDs from trisodium 4-sulfophthalic acid and ammonium molybdate. Furthermore, the framework structure of ZIF-8 coated with Au nanoparticles was modified on the outer surface of the Mn-CDs. This system effectively consumes glucose and converts it into a large amount of H₂O₂. Through the activity of the Mn-CDs, H₂O₂ was subsequently converted into oxygen, thereby alleviating hypoxia at the tumor site and promoting the photodynamic therapeutic effect of the gold nanoparticles [42]. Furthermore, some CDzymes not only possess an excellent POD-like catalytic activity but also have the characteristics of low toxicity and ease of surface modification. They can also be used to prepare functional composite materials. Song et al. coated CDzymes onto the surface of AuNPs to form an Au-CD nanocomposite with a carbon dot shell layer. Owing to the synergistic effect between AuNPs and the carbon dot shell, the catalytic activity can be tuned by irradiation with near-infrared light, which also results in an excellent surface-enhanced Raman activity, which can be used as a SERS monitor to achieve a sensitive and effective tumor catalytic therapy [43]. Sun et al. constructed a multifunctional therapeutic diagnostic nanocomposite (HSA-BFP@CDs) by conjugating trifunctionalized human serum albumin (HSA-BFP) as a therapeutic diagnostic agent targeting Aβ with CDs as ROS scavengers. When interacting with Aβ aggregates, HSA-BFP@CDs exhibit a fluorescence “on–off” effect at a wavelength of 700 nm, indicating their potential for detecting Aβ plaques and diagnosing AD early. Additionally, HSA-BFP@CDs effectively inhibited Aβ aggregation, increasing the survival rate of Aβ-treated cells from 74% to over 95%. Furthermore, HSA-BFP@CDs can scavenge various ROS, including •OH, O₂^−•, H₂O₂, and Aβ-Cu²⁺-induced ROS, alleviating cellular oxidative damage. Experiments using the Alzheimer’s disease model in Caenorhabditis elegans further demonstrated the multifunctionality of HSA-BFP@CDs in imaging amyloid plaques, alleviating oxidative stress, and reducing Aβ deposition in vivo, highlighting its potential for AD’s diagnosis and treatment by targeting Aβ and ROS [44].

3.4. Changes in Catalytic Conditions

In addition to the aforementioned method of regulating the catalytic performance of CDzymes by adjusting their own composition and structure, similar to natural enzymes, the catalytic activity of CDzymes is also greatly influenced by external factors such as light exposure, temperature, pH value, and substrate concentration [45]. Therefore, the catalytic activity can be regulated by adjusting the catalytic conditions. For example, Yin et al. reported that GQDs can effectively eliminate various free radicals, thereby protecting cells from oxidative damage. However, when exposed to blue light, GQDs exhibit excellent phototoxicity because of increasing ROS levels in cells, which is attributed to free radicals’ generation under light irradiation. They also confirmed that the formation of photo-induced ROS originates from electron-hole pairs. The photostimulated GQDs generate singlet oxygen through electron and energy transfer pathways. Under light exposure, GQDs facilitate the oxidation of nonenzymatic antioxidants and promote lipid peroxidation, thereby causing the phototoxicity of GQDs [46]. In addition to modulating enzyme-like activity through light, the POD-like activity of nitrogen-doped graphene quantum dots (N-GQDs) is strongly influenced by temperature, pH, and H₂O₂ concentration [47]. Compared with HRP, N-GQDs can exhibit a high catalytic activity within a wider pH range, but the catalytic activity significantly decreases when the pH exceeds 8.0. Similarly, N-GQDs can maintain a high catalytic activity between 25 °C and 45 °C, and the catalytic activity also significantly decreases above 45 °C. A colorimetric sensing system constructed under optimal conditions can be used for the convenient and sensitive determination of glucose.

4. Applications of CDzymes in the Treatment of Orthopedic Diseases

4.1. Osteoarthritis

Osteoarthritis (OA) is a type of degenerative joint disease that can lead to varying degrees of joint function impairment. Its main characteristics include degeneration of joint cartilage, thickening of the subchondral bone, formation of bone spurs, and joint degeneration [48]. The increased catabolism of the extracellular matrix (ECM) of joint cartilage is a key factor in the progression of OA. The aging of cells, increased expression of inflammatory factors, and oxidative stress associated with the limited regenerative capacity of tissues are all significant factors in the progression of OA. Generally, pathological deterioration is closely related to oxidative stress, which is caused by excessive ROS in the joint during OA. An increase in ROS aggravates the imbalance of joint microenvironment homeostasis induced by lipid peroxidation, DNA damage, and protein carbonylation [49]. Moreover, the excessive production of ROS also activates macrophages and stimulates proinflammatory signaling pathways, resulting in a more severe inflammatory response [50]. Hence, studies have focused on eliminating excessive ROS to relieve oxidative stress in the pathogenesis of OA, such as by using CDzymes as equivalents of natural antioxidant enzymes. Fang et al. prepared CDs from folic acid through the hydrothermal method shown in Figure 4a. These CDs rescue the oxidative stress, inflammatory response, extracellular matrix degradation, and cartilage degeneration induced by IL-1β. Moreover, CDs readjust the inflammatory and polarization states of macrophages induced by lipopolysaccharide (LPS) and inhibit the activation of the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways in chondrocytes under the influence of IL-1β, effectively delaying the onset of osteoarthritis [51]. Dong et al. incorporated cerium (Ce) and selenium (Se) into carbon dots to construct cerium–selenium-codoped carbon dots (CS-CDs) with a synergistic catalytic activity similar to that of SOD and GPx. In this self-assembled nanozyme system, the cerium active center catalyzes the generation of H₂O₂ by mimicking SOD, while the adjacent selenium components sequentially decompose H₂O₂ by mimicking GPx, thereby achieving an enhanced catalytic ability and excellent ROS-scavenging capacity. Carbon dots–carbon nanotubes (CS-CDs) were further loaded with Cy5-labeled anti-miRNA-155 to achieve macrophage subtype recognition through miRNA-155 quantitative imaging for the accurate monitoring of rheumatoid arthritis (RA) and to regulate the polarization of proinflammatory macrophages to achieve a synergistic RA improvement [52]. Additionally, the carbon dots themselves also have the ability to carry drugs. By loading drugs, they can achieve a synergistic therapeutic effect with drugs for the treatment of OA. Zhang et al. synthesized positively charged CDs with a porphyrin iron core and amino-functionalized surfaces to eliminate H₂O₂, superoxide anions, and hydroxyl free radicals simultaneously, as shown in Figure 4b. By using the Schiff base reaction, CDs loaded with MTX were combined with polyethylene glycol (CDs2-P@M), significantly prolonging their circulation time in the body. In a rat model of arthritis induced by collagen, CDs2-P@M accumulated at the affected joints, reducing the levels of ROS and inflammatory cytokines, readjusting the phenotype of macrophages, inhibiting the activation of osteoclasts, and significantly improving the symptoms of arthritis [53].

4.2. Osteoporosis

With the aging of the population and the extension of life expectancy, osteoporosis (OP) is increasingly becoming a global epidemic. OP is defined as a systemic bone disease whose clinical manifestations include decreased bone mass, deterioration of bone tissue microstructure, and increased bone fragility [54]. Generally, OP can be classified into two types according to its cause: primary and secondary osteoporosis [55]. Primary osteoporosis is mainly attributed to aging and a reduction in age-related sex hormones, whereas secondary osteoporosis is associated with adverse pathological conditions or drug treatments. However, unbalanced bone homeostasis always leads to enhanced bone resorption and reduced bone mass in patients with OP [56]. The asymptomatic and progressive nature of OP highlights the importance of the early identification of this disease. Although there are various treatment methods, prevention of the disease is the most important aspect of disease management. Currently, the treatment of osteoporosis is limited to antiresorption drugs that stimulate the formation of bone and anabolic drugs [57]. Owing to their obvious side effects, current treatments cannot achieve satisfactory therapeutic effects. Bisphosphonates are among the most widely used drugs in the treatment of OP, but they may cause serious bisphosphonate-related jaw necrosis, which can lead to severe pain in patients [58]. Jin et al. developed a new type of CD derived from alendronate (ALEN-CDs). ALEN-CDs exhibit excellent photoluminescence properties, biocompatibility, and bone-targeting capabilities. They can suppress the differentiation and maturation of osteoclasts, exert immunomodulatory effects by inhibiting the polarization of macrophages toward the proinflammatory M1 phenotype, and accelerate polarization toward the anti-inflammatory M2 phenotype. More importantly, the authors reported that, unlike alendronate, a high dose of ALEN-CDs does not cause bronchopulmonary osteonecrosis, which may be attributed to the regulation of M2 macrophage polarization. ALEN-CDs effectively reduce bone loss in ovariectomized (OVX) mice and improve osteoporosis symptoms [59]. In addition, regulating the local environment via excessive ROS has been regarded as a new strategy for restoring the intrinsic balance of bone metabolism. For example, Zhang et al. synthesized a new type of bioactive carbon quantum dot (CQD). It was also demonstrated for the first time that it has a significant anti-osteoporosis effect on the body. Unlike traditional antiosteoporosis drugs, CQDs have an excellent biocompatibility, and no adverse health effects are observed. Moreover, selectively inhibiting the activity of osteoclasts significantly increased bone mass and mechanical strength. This unique mechanism highlights the potential of carbon quantum dots as a breakthrough treatment option for osteoporosis, which can overcome the limitations and side effects of current treatment methods [60]. Additionally, iron overload increases the activity of osteoclasts and reduces the activity of osteoblasts, thereby causing osteoporosis. Li et al. constructed carboxymethyl chitosan-based carbon dots (AOCDs) as a calcium supplement to treat osteoporosis, which were found to act as antioxidants to alleviate osteoporosis caused by iron overload. Furthermore, by introducing calcium ions (AOCDs:Ca), the dual effects of antioxidation and calcium supplementation were achieved, significantly rescuing iron-induced osteoporosis in zebrafish. Importantly, AOCD:Ca effectively increased the bioavailability of calcium and achieved calcium supplementation at ultralow concentrations [61].

4.3. Osteomyelitis

Osteomyelitis (OM) is a bone inflammatory condition caused by infectious microorganisms, resulting in persistent local bone destruction and even necrosis [62]. OM can be classified into three types according to the source of infection: I, hematogenous OM; II, secondary OM (related to vascular or nerve dysfunction); and III, continuous infectious OM (associated with open fractures and orthopedic joint replacements). Excessive reactive oxygen and nitrogen species (RONs) can trigger inflammation without control and exacerbate the destruction of pulp tissue. Liao et al. proposed an effective antioxidant system (C-NZ/GelMA), which consists of CDzymes prepared from benzidine and methacrylated gelatin (GelMA). The antioxidant properties of CDzymes and the mechanical support of GelMA hydrogels are utilized to regulate the pulp inflammatory microenvironment and thereby promote pulp regeneration. This system can effectively eliminate ROS to restore the redox balance within cells and alleviate oxidative stress damage. Notably, it can also significantly enhance the polarization of regenerative M2 macrophages [63]. In addition, bacterial infection is the direct cause of OM and an important factor for the long-term nonhealing of OM. To develop effective treatment methods for bacterial infectious OM, researchers have endeavored to construct antibacterial platforms based on CDzymes. Zhou et al. synthesized carbon quantum dots (PL-CQDs) using ε-poly(L-lysine) as the raw material. The PL-CQDs possess antibacterial and osteogenic-promoting capabilities and are expected to enhance the therapeutic effect of OM. The PL-CQDs have no cytotoxicity and can kill methicillin-resistant Staphylococcus aureus and Escherichia coli. Moreover, the PL-CQDs promote cell migration and the osteogenic process. The transcriptome sequencing results revealed that the PL-CQDs significantly altered the extracellular matrix-receptor interaction signaling pathway and participated in various biological processes, such as the positive regulation of chondrocyte proliferation, the organization of collagen fibers, and the regulation of osteoblast proliferation. The immunohistochemistry indicates that PL-CQDs promote the repair of osteomyelitis through facilitating matrix deposition and vascularization at the bone defect site [64]. The role of nanomaterials is increasingly attracting the attention of researchers. The development of new antibacterial CDzymes can replace traditional antibiotics and achieve the goal of avoiding the risk of drug resistance. These findings provide more feasible and effective ideas for the clinical treatment of OM.

4.4. Intervertebral Disc Degeneration

Intervertebral disc degeneration (IVDD) is closely related to low back pain [65,66]. Although IVDD does not directly cause death, the pain, psychological burden, and economic burden it imposes on patients cannot be underestimated. Currently, the conservative treatment for IVDD can relieve pain, but it cannot reverse the progression of this disease [67]. Patients who cannot tolerate the pain usually adopt the strategy of the surgical removal of the degenerated intervertebral disc. However, surgical removal of the IVD affects the stability of adjacent intervertebral discs, and postoperative complications cannot be ignored. Increasing evidence indicates that the accumulation of ROS is closely related to the development of IVDD, as is inflammation [68]. Moreover, ROS play a relatively important role in many signaling pathways during the occurrence and development of diseases, and they can regulate IVDD by influencing inflammatory-related signaling pathways. In recent years, research on biomaterials used to regulate the inflammatory microenvironment for the repair of IVDD and its development has increased. The strategy of using CDzymes to target oxidative stress for the treatment of IVDD has attracted researchers’ attention. Wang’s group developed carbon dots derived from N-acetylcysteine (NAC-CDs), as shown in Figure 5a. The NAC-CDs have a good biocompatibility and strong SOD (250 U/mg⁻¹), CAT, and GPx-like activities and total antioxidant capacity and have strong free radical scavenging and antioxidant capabilities. They also maintain mitochondrial homeostasis and inhibit cell aging, thereby suppressing the expression of inflammatory factors in NPCs and significantly improving the magnetic resonance imaging grade, intervertebral disc height index, and histological score in a degenerative model [69]. Furthermore, to enhance the therapeutic effect, the authors coated Prussian blue nanozymes on the surface of CDzymes. Triphenylphosphine (TPP), a ligand that targets mitochondria, is used to modify CDs via Prussian blue (CD-PB) to remove excess ROS within cells and maintain NPCs in a normal redox state, as shown in Figure 5b. CD-PB-TPP can effectively prevent lysosomal phagocytosis, thereby achieving the efficient targeting of mitochondria. By exerting antioxidant enzyme-like activities, SOD and CAT significantly reduce ROS in mitochondria. CD-PB-TPP can rescue damaged mitochondrial functions and enable NPCs to recover from aging, catabolic metabolism, and inflammatory responses in vitro. In vivo assessment and histological analysis revealed that after a local application of CD-PB-TPP, the intervertebral disc height index, average gray value of the nucleus pulposus tissue, and histological morphology significantly improved in an intervertebral disc degenerative lesion model [70].

4.5. Malignant Bone Tumors

At present, the treatment options for malignant bone tumors mainly include surgery, radiotherapy, or chemotherapy [71,72]. The drawback of surgical removal is that the resection rate is low, whereas the recurrence rate is high. Moreover, many cancer patients are not suitable for surgical removal if their tumors have metastasized or showed other conditions. Moreover, radiotherapy and chemotherapy have significant side effects, a poor targeting ability, and high costs. Therefore, the current clinical treatment outcomes are not satisfactory. Compared with these conventional treatments, CDzymes have been widely applied in the field of tumor treatment due to their advantages: easy preparation, strong modification ability, few side effects, and good targeting ability. ROS have dual effects on the environment of malignant tumors. The level of ROS in the tumor microenvironment is generally greater than that in normal cells, which is conducive to promoting the expression of oncogenes, enabling cancer cells to proliferate and metastasize, and generating new blood vessels. If the level of ROS reaches a certain high level or even exceeds the threshold, it will cause apoptosis in the tumor cell environment, inhibiting the growth of tumor cells. Therefore, the current idea of applying the characteristics of CDzymes in the treatment of malignant tumors involves utilizing their photothermal/dynamic effects or exerting their POD-like activity to increase the level of ROS in the tumor microenvironment, causing excessive oxidative stress and thereby achieving the therapeutic goal of killing tumor cells. Wang et al. synthesized a novel CDzyme (Mn-CD) using toluene blue (TB) and manganese as raw materials. Mn-CDs achieved enhanced magnetic resonance (MR) imaging capabilities for the tumor microenvironment (acidic and glutathione). Moreover, the Mn-CDs exhibited efficient POD-like activity and catalyzed the conversion of H₂O₂ to hydroxyl radicals, which was significantly improved under light conditions. The results indicated that the Mn-CDs could achieve real-time MR imaging of the tumor microenvironment response through the aggregation of enhanced permeability and retention effects at the tumor site and could promote photodynamic therapy [73].

4.6. Bone Defect Repair

The incidence of postfracture infection is approximately 5%. It not only leads to prolonged nonhealing of the fracture but also causes serious conditions such as osteomyelitis [74]. Clinically, it is usually necessary to implant devices for fixation or filling the injured area to assist in bone repair. However, these implants may become “accomplices” for bacteria, creating conditions for the pathogens to hide, colonize, and cause an outbreak of infection. Recently, the treatment of infectious bone defects has faced limitations, such as poor vascularization of the grafts, antibiotic resistance, and long rehabilitation periods [75]. There is still a lack of comprehensive solutions that can activate multiple mechanisms to promote bone repair and achieve multiple strategies to kill bacteria simultaneously. In the microenvironment of infectious bone injury, an ideal material should possess antibacterial, osteogenic, and proangiogenic functions. Han et al. prepared arginine carbon dots (Arg-CDs) derived from arginine, which have shown extremely strong antibacterial and osteogenic activities. They further prepared a Schiff base bond between arginine–cyclodextrin (Arg-CD) and an aldehyde hyaluronic acid/gelatin methacryloxy (HG) hydrogel to respond to the acidic bone injury microenvironment and release Arg-CD. Free Arg-CD selectively generates excessive amounts of ROS to kill bacteria. Additionally, the HG composite hydrogel loaded with Arg-CD induced macrophage polarization toward the M2 phenotype by increasing the expression of interleukin-10 (Il10), resulting in excellent osteogenic activity. Arg-CD can endow the material with outstanding antibacterial and osteogenic activities, which is beneficial for the regeneration of infected bone [76]. Recently, they synthesized quercetin carbon dots (QCDs) through a hydrothermal reaction. Then, the QCDs were incorporated into a hydrogel of methyl acrylate-grafted amino boronic acid polyglutamic acid (PBA-m-PGA), thereby forming a composite hydrogel with ROS responsiveness. They reported that QCDs could activate the Rap1 pathway in bone marrow mesenchymal stem cells. On the one hand, they enhanced the osteogenic effect by promoting angiogenesis and the nuclear translocation of β-catenin. On the other hand, they promoted cell proliferation by activating the PI3K-AKT pathway. Their antibacterial mechanism involves the inhibition of the expression of key genes involved in bacterial cell wall synthesis (dapE), drug resistance-related genes (phoP), and energy metabolism genes (secA2). Their study not only elucidated the chemical structure of QCDs but also revealed that they could activate the Rap1 signaling pathway through interaction with RTKs, promoting bone regeneration while killing bacteria by destroying the bacterial cell wall and interfering with energy metabolism [77].

4.7. Periodontitis

Periodontitis is a common chronic oral disease worldwide. Its definition is the irreversible immune inflammatory destruction of the tissues that support teeth (including alveolar bone) [78,79]. An increasing number of studies have shown that the destruction of periodontal tissues is associated with a high level of ROS. Throughout the progression of periodontal disease, continuous inflammation triggers the excessive production of ROS by immune cells, which exceeds the antioxidant defense capacity of the cells. This excessive ROS induces oxidative damage, disrupts cellular metabolism, and leads to irreversible tissue damage. The subsequent chain reaction strengthens the infiltration of inflammatory cells and promotes the production of ROS and the release of inflammatory cytokines [80]. The antioxidant system typically hinders the ability of free radicals to extract electrons from healthy cells but instead provides electrons among free radicals, thereby effectively protecting healthy cells. Therefore, eliminating ROS to modify the periodontal microenvironment and reduce inflammatory responses may be an effective strategy for periodontal treatment. For example, Yu et al. developed the MT-CDzyme derived from melatonin. The MT-CDzyme effectively decreased the level of intracellular ROS, restored mitochondrial homeostasis, and inhibited the production of inflammatory mediators. Moreover, the results of the periodontitis mouse model demonstrated that the MT-CDzyme significantly suppressed the deterioration of alveolar bone and reduced osteoclast activation and inflammation, facilitating the regeneration of damaged tissues. Moreover, MT-CDs can eliminate free radicals, thereby regulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway to prevent cell damage and the generation of inflammatory factors [81]. In addition, bacterial infection is also an important component of periodontal diseases. Yang et al. reported copper-doped carbon dots (Cu-CDs). These Cu-CDs exhibit enhanced catalytic (CAT-like, POD-like) activities in the oral environment and are capable of inhibiting the attachment of initial bacteria (Streptococcus mutans), eliminating biofilms without affecting the surrounding oral tissues and without generating oxygen (O2) or ROS. In particular, Cu-CDs have a strong affinity for lipopolysaccharide and peptidoglycan, thereby endowing them with excellent antibacterial effects on Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), which can prevent wounds’ suppurative infection and promote rapid wound healing [82]. A summary of the application of CDzymes with different enzyme activities is presented in

Table 2. Synthesis and applications of different types of CDzymes.

Precursor	Synthesis	Enzyme-Like Activity	Application	Ref
Metal doping
NaFeEDTA	In situ pyrulysis	OXD (Vmax = 10.4 nMs−1, Km = 168 μM)	Phosphate monitoring	[16]
Mn, Mo	In situ pyrulysis	CAT	Photodynamic therapy	[42]
Cu, Zn chelated EDTA	In situ pyrulysis	CAT, SOD	Ischemia–reperfusion injury	[37]
Se	Hydrothermal	Antioxidant	Free radical scavenging	[21]
Mn (II) phthalocyanine	Hydrothermal	CAT	Antitumor	[32]
lignosulfonate Cu	Hydrothermal	OXD, POD, SOD, CAT	Wounding healing	[35]
Bi	Hydrothermal	CAT	Antitumor	[36]
Se, Ce	Hydrothermal	SOD, GPx	OA	[52]
PB	Hydrothermal	SOD, CAT	IVDD	[70]
Mn, toluidine blue	Hydrothermal	POD	Malignant bone tumors	[73]
Guanidine, Cu	Hydrothermal	POD, CAT	Periodontitis	[82]
Biomass
Scutellaria barbata and Herba Hedyotis diffusae	Hydrothermal	Antioxidant	Cigarette filter	[18]
Coffee	Hydrothermal	GSH oxidase	Antitumor Immunity	[15]
Quercetin	Hydrothermal	Antioxidant	Bone defect repair	[77]
Melatonin	Hydrothermal	Antioxidant	Periodontitis	[81]
Inorganic carbon
Graphite plates	Liquid-phase pulse method	Antioxidant	OP	[60]
Multiwalled carbon nanotubes	Oxidation reflux	POD (Vmax = 7.755 × 10−8 Ms−1, Km = 1.363 × 10−1 mM)	Glucose detection	[17]
Graphite powder	Oxidation reflux	POD	Detection of H2O2 and glucose	[47]
N-doping
p-Phenylenediamine	Hydrothermal	SOD, CAT	Anti-inflammation in liver	[20]
p-Phenylenediamine and Polyethyleneimine	Hydrothermal	Antioxidant	Wounding healing	[27]
o-Phenylenediamine	Hydrothermal	Antioxidant	OM	[63]
Tartaric acid and 3-aminophenol	Hydrothermal	OXD (Vmax = 121.95 × 10−8 Ms−1, Km = 0.61 mM)	Antitumor	[26]
L-glutamic acid, L-glutamine, succinic acid, citric acid, and glycine	In situ pyrulysis	POD		[38]
Folic acid	Hydrothermal	SOD	OA	[51]
Hemin chloride and polyethyleneimine	Hydrothermal	SOD, CAT	OA	[53]
Carboxymethyl chitosan and acrylamide	Microwave	Antioxidant	OP	[61]
S-doping
Thiourea	Hydrothermal	POD		[39]
Glutathione-	Hydrothermal	SOD, CAT, GPx	IVDD	[40]
Glutathione	Hydrothermal	SOD	Alzheimer’s disease	[44]
N-Acetylcysteine	Hydrothermal	SOD (250 U mg−1), CAT, PGx	IVDD	[69]
Thiourea	Microwave	Photo-oxidase (Vmax = 4.66 × 10−8 Ms−1, Km = 1.18 mM)	Antibacterial	[28]
P-doping
Alendronate	Hydrothermal	Antioxidant	OP	[59]

.

5. Opportunities and Challenges

Owing to the complexity of the disease mechanism and the special physiological environment of the affected area, traditional treatment methods for orthopedic diseases, such as surgical treatment and drug therapies, have a limited efficacy and cannot meet clinical requirements. Moreover, surgical treatment is prone to cause serious complications. Oral drug preparations exhibit poor pharmacokinetic characteristics, low bioavailability, and short half-lives. The systemic administration of some clinical drugs often leads to adverse reactions, such as anemia, leukopenia, and abnormal liver enzymes. Additionally, some patients develop drug resistance, and the therapeutic effect often weakens over time. New treatment methods urgently need to be explored. CDzymes are a new type of nanozyme featuring a small size, simple synthesis, high cost-effectiveness, flexible structure, excellent biocompatibility, and good photostability. Notably, CDzymes have a considerable specific surface area and abundant active groups, demonstrating a high catalytic activity. The development of new therapeutic applications of CDzymes in orthopedic diseases is highly important. Nevertheless, the rapid development of CDzymes presents both opportunities and challenges for the development of new alternative therapies for orthopedic diseases. The gradual application of CDzymes in the biomedical field of orthopedics still has many potential applications that need to be explored, and many difficulties need to be overcome. For example, the application of CDzymes in the sterile or septic loosening involved in joint replacement surgery shows great potential. The sterile loosening induced by wear particles is attributed to chronic inflammation originating from the interaction between implant wear debris and resident immune cells. However, septic loosening usually involves chronic infections related to the implant site and biofilms. For both sterile loosening and septic loosening, ROS cannot be ignored in the treatment process. CDzymes with a high ROS scavenging activity can be used as antioxidant catalysts to treat sterile loosening. In addition, some CDzymes produce highly toxic ROS to kill bacteria and destroy biofilms at the implant site, resulting in good POD- or OXD-like activities. These strategies involving CDzymes provide new insights for the treatment of orthopedic diseases.

Although the activity of CDzymes can be well regulated by various factors (such as size, doping, or surface modification), the rational design of CDzymes still requires a precise relationship between structure and catalytic function. For this purpose, interdisciplinary cooperative negotiations between experimental and computational research aim to explore the detailed catalytic mechanism of CDzymes in detail. The research by Yan is dedicated to explaining the potential mechanisms of the multiple enzymatic activities of CDzymes through density functional theory (DFT) studies. The theoretical principles obtained in their work can be screened through high-throughput calculations and reliably predict the SOD-like activity of CDzymes [83]. This also encourages us to transform the field of CDzymes from an empirical science to a theory–experimental combined science. Moreover, although an increasing number of studies have reported important discoveries of CDzymes in biomedical applications, there are several important overlooked aspects in clinical medical applications. Maintaining a balance between good therapeutic effects and potential biological safety is crucial. Owing to the different physical and chemical properties of CDzymes (such as size, surface charge, and hydrophilicity), the most prioritized consideration is to systematically evaluate their toxicity and stability in the blood circulation. When CDzymes are used as nanomedicines in the body, several important factors in pharmacodynamics studies need to be determined, including the therapeutic window, the maximum tolerated dose, and the minimum effective dose. Although Chen et al. reported that microorganisms in the gut microbiome use the pyruvate fermentation pathway to bind inorganic carbon from carbon nanomaterials to organic butyrate and that the resulting excess butyrate further affects the function (proliferation and differentiation) of intestinal stem cells [84], rigorous research on post-treatment evaluations should also be conducted, including the detailed pharmacokinetics, metabolism, absorption, biodistribution, and reversible (or even irreversible and delayed) toxicity of CDzymes.

6. Outlook

Since their discovery in 2007, nanozymes have become a research hotspot in the scientific community and have experienced rapid development. Over the past decade, CDzymes have been widely applied in the diagnosis and treatment of various diseases in various fields. This article provides only an overview of the progress of their application to diseases in the field of orthopedics. Notably, for researchers in related fields, fully understanding the synthesis principles and modification methods of CDzymes and scientifically applying them to appropriate disease models are important. Currently, the use of CDzymes is an emerging interdisciplinary field. From the design to the clinical application process, a large amount of basic research is needed to verify their comprehensive effects in multiple aspects. Similarly, CDzymes also face many ethical challenges. The approval of nanodrugs for clinical use still faces many difficulties and resistance. Although CDzymes are still at the primary experimental stage in many orthopedic disease treatment fields at present, we believe that with the continuous efforts of researchers and the continuous innovation of science and technology, CDzymes will eventually shift from the “backstage” of the laboratory to the “stage” of clinical practice in the near future, becoming an extremely important method for the treatment of orthopedic disease, increasing their benefits to human health.