IRON OXIDE NANOPARTICLES FOR DIAGNOSING AND TREATING DEGENERATIVE ORTHOPAEDIC DISEASES: A REVIEW

The prevalence of degenerative orthopaedic diseases, such as osteoarthritis, osteoporosis, and intervertebral disc disease, has increased due to the increasing prevalence and unsatisfactory therapeutic methods. Recently, different types of bioactive nanomaterials, such as iron oxide nanoparticles (IONPs), have raised much concern due to their ability to positively regulate the bone remodelling balance. Due to their magnetic characteristics, IONPs have been applied in magnetic resonance imaging in the clinic, but their ability to treat degenerative orthopaedic diseases has recently been shown both in vitro and in vivo . However, a comprehensive review of the potential utilization of IONPs in the orthopaedic field is lacking. Here, we summarize previous works in this review and discuss future research directions in this field.


Introduction
With the acceleration of ageing, the prevalence of degenerative orthopaedic diseases has increased rapidly, which has raised much concern.Degenerative orthopaedic diseases, which include many highly prevalent diseases, such as bone-featured osteoporosis (OP) and cartilagefeatured osteoarthritis (OA)/intervertebral disc disease (IDD), are characterized by senescence of bone and cartilage tissues.These diseases cause many elderly people to suffer, which is also the main cause of disability and death.
OP is a common degenerative orthopaedic disease seriously threaten the survival quality of elderly (Agrawal andGarg, 2023; Ensrud andCrandall, 2024).Researchers have reported that fragility fractures occur more often in OP patients, especially in the hip and centrum, which can cause long-term bedrest and eventually death.Although many drugs, such as bisphosphonates, denosumab and teriparatide, are available, their side effects and limited therapeutic effects are still unsatisfactory (Pant et al., 2023; Wei et al., 2022; Zheng et al., 2022).For example, in the treatment of OP, the patients with long-termed application of bisphosphonates might suffer from atypical fracture in the shaft of femur (Aouad et al., 2023; Black et al., 2020).The fracture is always defined as low energy damage due to the patients' own gravity, which might owing to the over-inhibition of bone resorption, which affects the bone remodeling for the trabeculae bone with appropriate micro-architectural structure for load-bearing (Kordoni et al., 2018; Lisnyansky et al., 2018; Saleh et al., 2013; Schilcher et al., 2011).
OA is also a common degenerative disease of the whole joint characterized by pathological cartilage damage, which is the most common cause of joint disability and pain in elderly individuals and affects more than 250 million people worldwide (Allen et al., 2022; Prieto-Alhambra et al., 2014).In the clinic, stepped therapeutic methods highlight the importance of pharmacotherapy.However, the drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs), glucosamine sulfate and sodium hyaluronate are always "safe but ineffective", which cannot intervene the www.ecmjournal.org European Cells and Materials Vol.48 2024 (pages 45-65) DOI: 10.22203/eCM.v048a04process of diseases, which makes it difficult to achieve a satisfactory therapeutic effect (Eckstein et al., 2006; Ibrahiem et al., 2023; Liu et al., 2021; Moseng et al., 2024; Pei et al., 2023; Shavlovskaya et al., 2023; Wu et al., 2023).Those patients can barely avoid receiving the surgical treatment due to the progression of diseases and the diseasemodifying drugs is still in development (Li et al., 2023; Makarczyk et al., 2021; Shavlovskaya et al., 2023).The situation of IDD is similar to that of OA.Hence, the novel pharmacological therapeutic method is in urgent need for those degenerative orthopedic diseases.
Recently, with the development of nanotechnology, the treatment of degenerative orthopaedic diseases has become possible.These nanomaterials easily pass through physiological barriers due to their unique physicochemical properties, such as a high surface-to-volume ratio, which allows them to be applied in drug delivery and enhances their therapeutic effect (Bartkowski et al., 2024; Liang et al., 2023b).As a typical nanomaterial, iron oxide nanoparticles (IONPs), including Fe 3 O 4 , y-Fe 2 O 3 , and hybrid ferrites, have garnered much attention.These nanoparticles can carry iron, which can be detected by existed medical device such as magnetic resonance imaging (MRI).Also, it can induce biological activities due to the releasion of iron ions and the properties of nanoparticles.In previous translational research, IONPs exhibited excellent biocompatibility and biodegradability in vivo (Paik et al., 2015; Patil et al., 2015).Additionally, they are sensitive to magnetic fields, enabling their use in both diagnostic and therapeutic approaches, especially for the cellular and subcellular diagnosis (Kim et al., 2020; Shi et al., 2020; Vallabani et al., 2019; Wu et al., 2015).Meanwhile, due to the modifiable property, the IONPs have been used to prepare a lot of multi-functional agents with the integration of diagnosis and treatment have been developed, especially in the oncological field.
Although the IONPs have been maturely applied in the biomedical field, the research is in its infancy in orthopedic field.According to previous reports, IONPs has been explored to be applied in the diagnosis and treatment of different orthopedic diseases, such as OA, OP, bone tumors, fractures, osteolysis, and bone defects (Fig. 1) (Guo et al., 2020; Pang et al., 2021; Sadeghzadeh et al., 2023; Vergnaud et al., 2022; Wang et al., 2021a; Zhang et al., 2024).The nanoparticles exhibited great bioactivity when locally administrated in bone tissues alone or combined with other motifs, such as the enhancement of osteogenic and chondrogenic differentiation.Also, the unique magnetic properties make the IONPs could be used for magnetic-respond drug/stem cell tracing and delivery in orthopedic diseases (Chang et al., 2021; Xia et al., 2019b).When combined with the intervention of external magnetic field, the IONPs might translate the magnetic into other stimulation via different electromagnetic effect, such as the magnetocaloric effect, magneto-force effect (Del Bianco et al., 2022; Hamad et al., 2021; Ma et al., 2022; Wang et al., 2021b).The physical intervention is especially emphasized in the abundance of bone matrix, which finally act on the cells and induced the alteration in the cellular behaviors, such as the regulation of osteogenic and chondrogenic differentiation (Harris et al., 2015).
Although the IONPs is a promising agent due to the magnetic properties and biological activity, the biosafety and efficiency in diagnosing and treating degenerative orthopedic diseases are still worrying.As is known, the iron ions are tightly related to the ferroptosis, which has been regarded as the key pathological process in the inflammation and senescence (Coradduzza et al., 2023; Fan et al., 2024; Sun et al., 2024).In the oncological field, the ferroptosis is an effective weapon for killing the tumor cells.However, in the treatment of degenerative diseases, it might be regarded as a "double-edged sword", which might contribute to the acceleration of pathological process.Also, too many kinds of IONPs have been developed for the biomedical application due to the excellent extensibility.However, how to use an appropriate IONPs agent to intervene the determined pathological process in the degenerative orthopedic disease is still unclear.Briefly, a retrospective review based on the translational application perspective in orthopaedic field is still in lack.Herein, we summarize the application of IONPs in diagnosing and treating degenerative orthopaedic diseases, which might provide new insights for future research in this field.

The Preparation of IONPs
IONPs can be prepared by various methods, including physical, chemical and biological methods, which is tightly linked with the physicochemical properties (Laurent et al., 2008; Wu et al., 2015).Due to the lack of ability to precisely control the size of IONPs in the nanoscale, the physical approachs, such as the electron beam litography, aerosol, gase phase deposition, and powder ball milling, are not widely applied (Ling et al., 2015).The biological process relies on the redox reaction, which is more promising to be explored in the future.We considered that the in-situ synthesis of IONPs in vivo might be a ultimate aim of the biological method.However, the present application is still limited, and the application of physical and biological methods account for less than 10% in the preparation of IONPs (Ali et al., 2016; Revathy et al., 2023; Samrot et al., 2021).Hence, we focus on introducing the chemical synthesis routes.
The co-precipitation method is among the most simple and efficient synthesis procedures, which involves the mixing of multiple metal salts and precipitants to trigger a reaction, ultimately leading to the formation of a mixture comprising iron oxide and metal ions in the reaction solution (Sharouf andSaffour, 2024; Wahfiudin et al., 2024).The adjustment of factors such as pH value, reaction time, precipitant type, and solution concentration en-European Cells and Materials Vol.48 2024 (pages 45-65) DOI: 10.22203/eCM.v048a04ables the achievement of control over product particle size and composition (Wahfiudin et al., 2024).Nevertheless, this method is associated with a broad particle size distribution and challenges in precisely controlling product composition.To overcome these challenges, the improved co-precipitation techniques with the assistance of magnetic field, ultrasound, or functionalized bases have been developed (Liu et al., 2011; Pereira et al., 2012; Petcharoen and Sirivat, 2012; Remya et al., 2016; Roy et al., 2016; Suh et al., 2012; Wu et al., 2011).
To precisely control the size and shape of IONPs, the solvothermal method, which is also named as thermal decomposition, was developed (Hufschmid et al., 2015; Park et al., 2004).The method entails the heating of a solution comprising a mixture of metal ions and surfactants to yield IONPs with tunable particle sizes.By manipulating parameters such as temperature, concentration, surfactant type, and heating duration, this method allows for the precise attainment of desired product particle sizes and shape (Sharifi et al., 2012).However, this approach necessitates high temperatures and a complex preparation process, which make it not environmentally friendly.Also, the application of toxic chemicals in the synthesis processes make its own a risk in biocompatibility to be directly applied in the medical scene (Roy et al., 2021).
The sol-gel method is also a common method for the preparation of IONPs, which involves the swelling of metal ions in an organic solvent, followed by hydrolysis and repolymerization to form an iron oxide gel system (Darmawan et al., 2010; Puscasu et al., 2016).Subsequent heat treatment leads to the production of nanoparticles.This approach exhibits high precision and controllability, enabling the regulation of product morphology and crystal form through the manipulation of conditions such as hydrolyzing agent type and concentration (López-Sánchez et al., 2022; Panda et al., 2024; Waqas et al., 2024).However, it necessitates a prolonged preparation time, a complex process, and may result in environmental pollution due to the use of organic solvents.
Besides, a lot of methods such as microemulsion, hydrothermal, sonochemical and electrochemical deposition methods were employed for the preparation of IONPs.The microemulsion method used two immiscible liquids to form a confined environment for the nucleation and controlled growth of nano-and micro-particles (Hwang, 2024; Morán et al., 2023; Rahman et al., 2024).Although the size of the IONPs could be easily controlled by regulating the micelles, the limited crystallinity and yield make it hard to be translated in clinic use (Chaudhari and Panda, 2023).The hydrothermal method is relatively low-cost and easy to be performed.However, the final size of particles is not easy to be controlled (Hang et al., 2024; Ta et al., 2024).We have listed the advantages and disadvantages of different methods in the preparation methods with ability to balance the yield and the ability to control the size and shape of IONPs is still in need to be developed.

The Biocompatibility of IONPs
When IONPs are applied in biomedical applications, biosecurity, which includes adverse effects when animals are exposed to nanoparticles, is highly important.Thus, we used a single section to exhibit the biocompatibility of IONPs before the application in orthopedic field.A previous review reported the therapeutic efficacy, migration and metabolism of IONPs in vivo (Malhotra et al., 2020).Although IONPs have been used in different ways in the biomedical field, toxicity is still a challenge that hinders their clinical translation.Although many types of IONPs exist, they have similar structures, including iron oxide cores, polymer coatings, and external layers.Previous work has shown that the toxicity of IONPs is mostly dependent on the shape and size of the nanoparticles and the presence of coating materials (Hussain et al., 2005; Jeng and Swanson, 2006; Karlsson et al., 2008; Karlsson et al., 2009; Malehmir et al., 2023).Herein, we separately introduce the aspects for the reference to prepare more bio-friendly IONPs for treating degenerative orthopaedic diseases.
The size of IONPs is an important factor affecting the cellular toxicity, which is mainly related to metabolism in vivo.In general, smaller IONPs (<10 nm) are metabolized by renal extravasation, while larger IONPs (>200 nm) are captured by the spleen, which indicates that IONPs with a size of 10-100 nm are more suitable for application in vivo (Choi et al., 2007; Gupta andGupta, 2005).Additionally, many studies have shown that different shapes of IONPs are linked to toxicity.For example, spherical IONPs have been observed to have lower toxicity than other shapes, while rod-shaped IONPs have been reported to have greater toxicity (Ran et al., 2015).
Previously, it was reported that a polymer coating is the most important structure for relieving the toxicity of iron oxide and could also prevent the aggregation of nanoparticles (Gupta and Gupta, 2005).The possible potential mechanisms include enhancing the stability of IONPs and reducing the speed of iron ion release from the iron oxide core.Previous results have shown that the albumin nanoparticle coating provides a stable biocompatible shell and prevents the cytotoxicity of magnetite nuclei.After prolonged exposure (48 hours), IONPs become cytotoxic due to the produc-tion of free radicals, but this toxic effect can be neutralized by the use of polyethylene glycol (Abakumov et al., 2018).Previously, we found that polymer coatings could relieve iron overload-induced OP by scavenging reactive oxygen species (ROS) (Yu et al., 2020).As is widely known, the ferroptosis is a kind of cellular death based on the lipid peroxidation and the generation of ROS, which means that the ROS-scavenging polymers are also promising in reducing the toxicity by intervene the cellular behavior (Cao et al., 2024; Endale et al., 2023; Teschke, 2024).So, a lot of polymer materials with excellent antioxidant properties is also promising to be used for the synthesis of novel IONPs with more excellent biocompibility and diversified bioactivity, and the area is worth to be explored in the future.Each coating material has its own advantages and disadvantages, and we should pay attention to the selection (Abakumov et al., 2018).However, studies on the relationship between coating materials and toxicity in different microenvironments are still limited, and more studies need to be conducted.
The biosafety of IONPs is a crucial aspect that should be thoroughly examined and addressed for their successful clinical translation (Wang et al., 2024a; Yang et al., 2023).While IONPs offer immense potential in various biomedical applications, such as drug delivery, magnetic resonance imaging, and hyperthermia treatment, their longterm safety profile remains a significant concern (Marycz et al., 2020; Moacă et al., 2023).IONPs are typically designed to degrade over time, releasing iron ions that are subsequently metabolized by the body.However, uncontrolled degradation can lead to excessive accumulation of iron, potentially causing oxidative stress and cellular damage.Therefore, strategies to precisely regulate the degradation rate of IONPs are crucial.As is shown in Fig. 2, a work used a continuous flow system to unveil the biologically degradation behavior in vivo and characterize the degraded products, which is important in promoting the clinical application of IONPs (Yang et al., 2024).The improvement of biosafety can be realized by the application of novel coatings or modifications that stabilize the nanoparticles for a desired duration, ensuring that they degrade only when needed (Gu et al., 2024; Natarajan and Tomich, 2020; Zhong et al., 2019).Moreover, the use of targeting ligands that specifically deliver IONPs to target cells while minimizing accumulation in healthy tissues could also be explored (Israel et al., 2020; Liao et al., 2015; Park et al., 2008; Riegler et al., 2013; Zhi et al., 2020; Zhou et al.,  2022).The biosafety of IONPs is a multifaceted issue that requires a comprehensive approach to address.By controlling the degradation rate, avoiding long-term accumulation, and mitigating potential toxic side effects, researchers can enhance the safety profile of IONPs and pave the way for their successful clinical translation.

Magnetic Nanoparticles for Imaging Innovations
In enhanced Magnetic Resonance (MR) detection, the most widely applied contrast agent is gadolinium (III).However, its high toxicity, especially in patients with liver and kidney failure, has raised significant concerns and limited its clinical application (Chen et al., 2011; Di Marco et al., 2017).In previous work, ultra-superparamagnetic iron oxide nanoparticles (SPIONs) were shown to significantly reduce the transverse relaxation time (T2) during MRI (Ajayi et al., 2023), and these materials have the potential to be applied as novel contrast agents.Recently, the imaging properties of newly prepared IONPs have been shown to be similar to those of gadolinium-based contrast agents, which are in the process of clinical translation (Liu et al., 2013) IONPs can be used for MRI of different tissues.Usually, nanoparticles less than 10 nm in size are excreted in the urine, while a large number of nanoparticles larger than 200 nm are engulfed by the digestive system.This phenomenon indicated that the IONPs used for imaging different organs need to be of different sizes and shapes.Larger IONPs accumulate in the reticuloendothelial system (RES), which enables the imaging of the liver and spleen (Dadfar et al., 2019).Moreover, particles with sizes between 20 and 150 nm tend to deposit in connective tissues (bone, tendons, and muscles), stomach, and kidneys, as reported previously (Wang et al., 2022a).As mentioned above, the size of IONPs could be easily controlled by adjusting the preparation process.Determining the relationship between the size of IONPs and their enrichment in organs is important for future research and is highly valuable for preparing individual products for organ imaging.
In bone tissue imaging, imaging depth is still a challenge that limits imaging technology, but IONPs might provide a possible solution.Recently, magnetic particle imaging (MPI) technology, which aims to evaluate the electromagnetic properties of IONPs according to the gradient relationship between the magnetic field and concentration of IONPs, has been developed.IONPs saturate according to the direction of the magnetic gradient, except in a field without a magnetic field (Graeser et al., 2019; Panagiotopoulos et al., 2015; Saritas et al., 2013).The oscillating be-European Cells and Materials Vol.48 2024 (pages 45-65) DOI: 10.22203/eCM.v048a04haviours of IONPs make it possible to visualize and quantify the imaging results, which eliminates common issues in optical imaging, such as the disturbance of autofluorescence and signal attenuation in tissues (Bulte et al., 2015; Saritas et al., 2013).Moreover, MPI technology is not dependent on negative contrast for screening IONPs, which could avoid confusion in visualization at the air medium and tissue interfaces (Talebloo et al., 2020).Moreover, MPI enables continuous longitudinal screening of signals in samples, which is vital for cell tracing in vivo (Kang et al., 2014; Rana et al., 2010).Previously, the IONPs conjugated with collagen-binding peptides and IONPs can be detected via MRI methods for the diagnosis and treatment of osteoarthritic joints (Guan et al., 2023).We hope similar studies will contribute to the diagnosis of the pathological process of orthropedic diseases.

Application of IONPs in the Treatment of OA
OA is a representative degenerative orthopaedic disease characterized by the deterioration of cartilage and subchondral bone (Abdelbari et al., 2023).Cartilage tissues are regarded as avascular connective tissue without a self-renewal ability, but cartilage regeneration has become a challenge (Bullock et al., 2018).Herein, we investigated the application of IONPs in the treatment of OA and in the treatment of cartilage-derived degenerative orthopaedic diseases, including as nanocarriers and diagnostic/therapeutic agents.
Due to the absence of the vasculature, the entry of agents into the joint carve has been regarded as a complex problem.Methods to deliver drugs into the joint carve have raised much concern.The systemic administration of drugs is limited by systemic side effects and low bioavailability (Abbas et al., 2022b; Mosallam et al., 2022; Yang et al., 2011).Local intra-articular injection is a promising method for directly delivering bioactive agents to cartilage lesions.However, repeated punctures increase the risk of iatrogenic infection.Nanocarriers have been developed for efficient drug delivery via the joint route to address these challenges, and they improve drug delivery efficiency while minimizing adverse effects on other organs and tissues (Abbas et al., 2022a; Mohamed et al., 2020).Magnetic targeting combined with IONPs has been shown to be efficient for drug delivery (Ong et al., 2020, Suryadevara et al., 2023).IONPs, which are easy to fabricate, play a vital role in the controlled delivery of bioactive agents to the determined area and have shown advantages in terms of biocompatibility and surface functionalization (Abbas et al., 2022a, Das et al., 2019, Son et al., 2015).Molecular drugs can easily bind directly to the iron oxide surface for local delivery (Ibrahiem et al., 2023; Partain et al., 2020).In addition, Mei et al. (2016) prepared IONPs with superparamagnetic properties using a high-temperature thermal decomposition method and subsequently coated them with PEG.The modified IONPs had a particle size of 5.9 ± 1.1 nm and could easily penetrate into cartilage for drug delivery.Additionally, other works have used WYRGRL, which is a short peptide that combines with COL2A1 in the extracellular matrix of cartilage (Papadimitriou et al., 2014; Yarmola et al., 2016).Also, the IONPs conjugated with C5-24 peptides increased the retention of hyline cartilage when administrated in OA knees in a previous study (Guan et al., 2023).Additionally, IONPs contribute to cartilage repair and regeneration by recruiting bone mesenchymal stem cells (BMSCs) to specific locations and promoting their expression while causing fewer inflammatory responses (Yang et al., 2019a andYang et al., 2019b).These innovative approaches present promising avenues for advancing OA treatment.
In addition to drug delivery, IONPs have also been widely used directly for diagnosing and treating OA (Fig. 3).The use of COL2A1-targeting IONPs can aid in the use of MRI to distinguish cartilage with early degenerative characteristics from healthy cartilage to achieve molecularlevel diagnosis (Wu et al., 2023).Additionally, chitosanmodified IONPs have been shown to be efficient at labelling cells without altering their differentiation ability, which can be useful for intra-articular imaging.Interestingly, a team used IONPs to collect CTX-II, an important biomarker in osteoarthritic joint carves, which is called "magnetic capture" and is helpful for the early-stage diagnosis of OA (Garraud et al., 2016; Yarmola et al., 2016).As metal oxide nanoparticles, IONPs were proven to significantly improve histopathological damage to rat knee joints by regulating OPG, RANKL, ERK1, and MAPK levels (Ibrahiem et al., 2023).In addition, IONP-containing biomaterials, such as diphasic magnetic nanocomposite scaffolds, nanovehicles, and PLGA microspheres, have been developed for the treatment of OA (Butoescu et al., 2009; Huang et al., 2018; Zhang et al., 2020; Wang et al., 2024b).In vitro experiments have shown that IONPs can promote the differentiation of BMSCs into chondrocytes and upregulate the Ihh/PTHrP signaling pathway, providing a potential therapeutic approach for treating cartilage degeneration-related diseases (Jiang et al., 2017).Additionally, studies have explored the effects of IONPs on the chondrogenic differentiation of human bone marrow stromal cells (HBMSCs), neonatal, and adult chondrocytes.It was found that the viability of all cell types was unaffected; however, the cell morphology shifted to a "stretched" phenotype after SPIO uptake, and the proliferation of neonatal chondrocytes decreased after SPIO uptake (Saha et al., 2013).In vivo studies have found that 12.75 µg/mL M-SPIO can successfully label human articular cartilage-derived chondroprogenitor cells with minimal impact on cell viability, MSC marker expression, and differentiation potential (Vinod et al., 2019).It also does not affect the production of major cartilage matrix components (Ramaswamy et al., 2009).However, the potential regulatory mechanism has not been elucidated well, and whether the therapeutic effects are due to nanopar- ticle properties or degradation products remains unclear and is worth exploring (Table 2).
In addition to the application of IONPs as drugs to treat OA, IONPs are more likely to be applied in combination with stem cells or chondrocytes in the cell tracing field and can exhibit therapeutic effects.IONPs combined with MRI T2 imaging can maintain the stemness of adipose-derived stem cells (ADSCs), which is promising for application in MRI-assisted cartilage tissue engineering (Xie et al., 2019).Naosuke Kamei et al. prepared a cell delivery system to deliver mesenchymal stem cells (MSCs) to the cartilage defect area for cartilage repair (Kobayashi et al., 2008).They subsequently conducted clinical work and reported that IONPlabelled autologous MSCs were safe for repairing cartilage defects in the knee, while newly formed cartilage was observed 48 weeks after surgery (Kamei et al., 2018).Additionally, IONP-labelled chondrocytes can also be guided by a magnetic field to the cartilage defect area for cartilage repair (Gong et al., 2018).As the autologous chondrocyte implantation (ACI) technique has been regarded as effective for treating cartilage defects, the use of IONP-labelled chondrocytes is similar to that of chondrocyte transplantation, which seems to have the advantage of minimal invasion.Briefly, IONPs combined with stem cells or chondrocytes are easy to translate in the clinic.However, long-term studies on the safety and effectiveness of these treatments are still needed.

Application of IONPs in the Treatment of OP
OP is a common bone degenerative disease with an extremely high prevalence in elderly individuals.OP can be divided into two subtypes: decreased bone formation activity and increased bone resorption activity (Compston et al., 2019; Ensrud andCrandall, 2024).Previously, IONPs were shown to be effective in regulating the behaviour of osteoblasts and osteoclasts.Chitosan-coated IONPs modified with chitosan and hydroxyapatite were reported to be effective at enhancing the proliferation of osteoblasts while protecting the cells from exogenous stimuli and promoting osteogenic differentiation (Shi et al., 2012; Tran et al., 2012; Tran and Webster, 2011).Similar osteogenicenhanced effects of IONPs were found in osteoporotic fracture models, while implant osseointegration was significantly enhanced (Anjum et al., 2023; Fouad-Elhady et al., 2020; Hedvičáková et al., 2023, Paun et al., 2018).Moreover, with the assistance of a magnetic field, IONPs can be guided to any bone area needed to enhance the formation of bone tissue, which could reverse the process of OP (Tran and Webster, 2013).In the same period from approximately 2012-2015, bone-targeting IONPs were developed for the radiological evaluation of bone metabolic activity (Panahifar et al., 2013).Additionally, the inhibitory effect of IONPs on osteoclasts was reported to be dependent on the TRAF6-p62-CYLD pathway (Li et al., 2021; Liu et al., 2019).However, with the increased awareness of iron overload-induced OP, the application of IONPs in treating this disease is controversial.In 2020, we reported that IONPs coated with antioxidative polysaccharides can release iron ions in bone mass without causing iron accumulation-related OP, which might be explained by the ROS scavenging effect of polysaccharides (Yu et al., 2020).Subsequently, IONPs combined with typical anti-OP drugs, such as bisphosphonates, were prepared for the treatment of OP in our and other works (Lee et al., 2016; Zheng et al., 2022, Panseri et al., 2012)  which is beneficial for bone renewal and the function of bisphosphonates (Fig. 4).IONPs can be combined with different materials, such as calcium phosphate cement (CPC) (Xia et al., 2019a), or synthesized into mesoporous silicacoated magnetic (Fe 3 O 4 ) nanoparticles (M-MSNs) (Jia et al., 2019), both of which enhance the osteogenic activity of stem cells through the WNT/β-catenin signaling pathway.Marycz K et al. (2020) synthesized the α-Fe 2 O 3 /γ-Fe 2 O 3 nanocomposite does not induce an immune response and regulates integrin expression; it also enhances the osteogenic differentiation of osteoblasts and triggers apoptosis of osteoclasts.The combined use of a 1-2 T static magnetic field (SMF) and IONPs reduces iron uptake by osteoclasts and decreases oxidative stress levels during osteoclast differentiation.At the molecular level, the 1-2 T SMF combined with IONPs inhibits the expression of the NF-κB and MAPK signaling pathways (Zhang et al., 2022).Meanwhile, the appropriate ratio of Fe/Ca = 1:15, mol/mol (SPIO@15HA) inhibited the formation of osteoclasts through the TRAF6-p62-CYLD pathway.Besides, the osteogenic differentiation process was enhanced by the regulation of TGF-β, PI3K-AKT, and calcium signaling pathways.Furthermore, the overexpressed cytokines such as OPG, CSF2, and CCL2 also contributed to the maintainence of bone remodeling balance (Li et al., 2021).The senescence-associated secretory phenotype of immune cells and bone-related cells is widely recognized as an important source of inflammatory cytokines that contribute greatly to the progression of OP.Whether IONPs have a regulatory effect on the production of inflammation still needs to be explored.

Application of IONPs in Intervertebral Disc Diseases
The intervertebral disc is another site that is linked to orthopaedic degenerative diseases and is also a leading cause of disability in elderly individuals, and the common clinical manifestation is low back pain (Xin et al., 2022).The exploration of IONPs in intervertebral discs has focused mainly on diagnosis or combination with stem cell therapy for IDD.For example, Guillaume Bierry et al. (2009) used IONP-enhanced MRI to identify infectious and degenerative vertebral disorders, which could distinguish between the two diseases according to the quantitative results.In other studies, IONPs were used to mark MSCs to evaluate their survival and differentiation (Handley et al., 2015; Hang et al., 2017; Saldanha et al., 2008).Additionally, recent research has used IONPs as a magnetofection system to deliver miR-21 into stem cells for intervertebral fusion operations, which is the only therapeutic application of IONPs in IDD (Wang et al., 2023a).Studies have shown that inflammation, mitochondrial DNA damage and apoptosis play an important role in the pathological process of intervertebral disc cell degeneration (Zhou et al., 2024).Silence-activated transcription factor 3 (ATF3) blocks the pathological process of IVDD by regulating iron apoptosis, apoptosis, inflammation, and extracellular matrix (ECM) metabolism in nucleus pulposus cells (NPCs) (Wang et al., 2024c).Sirtuins/SIRTs and their related activators regulate autophagy, myeloid apoptosis, oxidative stress and extracellular matrix degradation, and have positive effects on the treatment of IVDD (Shen et al., 2024).Prolonged exposure to high concentrations of IONPs may induce oxidative stress and inflammatory translation (Vidya Balakrishnan et al., 2024), but there is insufficient evidence of INOPs and inflammatory response in the treatment and imaging of IDD.Overall, the application of IONPs for treating degenerative diseases of the spine is still in the initial stages.Based on the similar pathogenesis of IDD and OA, future work on the spine is warranted.

Discussion
In the field of biomaterials, nanoparticles have been widely applied due to their unique physicochemical properties (Zheng et al., 2024).These nanoparticles can be widely used for diagnosing, treating and preventing orthopaedic diseases.Herein, we chose IONPs as representative nanoparticles and summarized their application in the orthopaedic field.Compared with other types of nanoparticles, IONPs have the unique advantage of magnetic properties and are also an important trace element in the human body.Iron metabolism in humans is a complex process involving duodenal enterocytes, plasma, erythroid bone marrow, the spleen, and the liver (Chifman et al., 2014).Many degenerative diseases have been linked to ferroptosis, which is a unique type of cell death mediated by iron-dependent lipid superoxidation (Jiang et al., 2021).Thus, the biosecurity of IONPs has been challenged.However, in our and others' previous work, suitable polymer coatings that can scavenge the existing ROS produced during ferroptosis can help to prevent iron accumulationinduced degenerative diseases (Guan et al., 2023; Yu et al., 2020; Zheng et al., 2022).Recently, many biosynthetic polymers with antioxidant activity, such as lignin, chitosan, and functional lipidosomes, have been identified (Pei et al., 2023; Zheng et al., 2021a; Zheng et al., 2021b).Surprisingly, we also found that IONPs can prevent the senescent secretion phenotype (data not published), which means that the intracellular delivery of Fe 2+ might involve an independent pathway to improve the inflammatory microenvironment.The crosstalk between IONPs and the immune system is worth exploring.
In addition to their direct effects, the synergistic effects of IONPs with typical therapeutic methods attracted our attention.The most common application is the combination of IONPs with stem cells, in which the IONPs play a multimodal role.In the process of clinical translation of stem cell therapy, the greatest obstacle is the risk of tumorigenicity, while the IONPs can act as surveillants.In preclinical and clinical experiments, IONPs can be used to collect data for the analysis of the proliferation and differentiation of stem cells.On the other hand, the IONPs could directly act on stem cells and regulate their behaviour, enhancing their ability to promote osteogenesis/chondrogenesis for tissue repair to treat degenerative orthopaedic diseases (Table 3).In addition to the advantage of integration of IONPs with stem cells, several distinct challenges still remained to be addressed.A major challenge lies in achieving highefficiency labeling of stem cells with IONPs while maintaining cell viability and functionality (Berman et al., 2011; Küstermann et al., 2008; Lin et al., 2017; Zheng et al., 2016).It was reported that the surface modified IONPs performed better in the long-term trancing of stem cells, such as the application of glucosamine, self-assembled peptide amphiphile, high-molecular polymers, amine and silica (Guldris et al., 2017; Yang et al., 2016; Yao et al., 2020; Liang et al., 2023a, Liu et al., 2020).Current methods often result in variable labeling efficiencies, which can limit the accuracy of tracking and monitoring stem cells, which could be improved by the development of device to generate focusing magnetic field and IONPs with more precise magnetic properties (Liu and Ho, 2017; Wang et al., 2020; Wang et al., 2022b).faces numerous hurdles, including concerns regarding biocompatibility, long-term safety, and regulatory approval.
IONPs exhibit significant potential when integrated with drugs, genes, and biomaterials to form multifunctional nanoplatforms aimed at synergistic therapeutic outcomes.As drug carriers, IONPs facilitate precise delivery via magnetic targeting, enhancing bioavailability and therapeutic efficacy.They also function as sustained-release agents, extending drug release and therapeutic duration (Tran et al., 2022).The multifunctional hybrids are used to treat cancers and achieved a lot of success (Ghadimi Darsajini et al., 2023; Feng et al., 2023; Hasani et al., 2023; Wang et al., 2023b).However, in the orthopedic field, the application of multifunctional IONPs containing hybrids in diagnosing and treating diseases is still in the early stage.Previously, the combination application of IONPs with different drugs, such as salicylic acid, dexamethasone, folic acid, alendronate, and exosomes were proven efficient in treating OA and OP (Ibrahiem et al., 2023; Marycz et al., 2020; Shah et al., 2017; Tran et al., 2022, Marycz et al., 2022, Meng et al., 2013).However, current work are more likely to use the IONPs as a drug which can synergetically perform therapeutic effect with the typical drugs.As we previous reported, the multifunctional nanoplatforms can be obtained as two styles: the multifunction of one single functional unit, and the combination of different functional units (Zheng et al., 2024).Due to the complex structure and physicochemical properties, IONPs are easily to be used as therapeutic core and nano-vechiles which is easily to be translated to treat diseases with different pathologies, such as OA.However, various factors, such as charge, particle size, and surface modifications, influence the nanoplatform preparation methods based on IONPs.Also, drug-IONPs integration may alter physicochemical properties, affecting stability and bioactivity.The integration process requires careful consideration of compatibility, stability, biological activity, and safety of the nanoplatforms.
In conclusion, IONPs are novel nanoagents that could be translated to the clinic for the treatment and diagnosis of orthopaedic degenerative diseases.However, some unsolved questions worth studying persist, such as the improvement of biosecurity and potential molecular mechanisms involved in regulating cellular behaviours.After these obstacles are solved, more clinical trials are needed European Cells and Materials Vol.48 2024 (pages 45-65) DOI: 10.22203/eCM.v048a04 in the future.

Fig. 2 .
Fig. 2. A flow system which can dynamically monitor the degradation behavior of IONPs in vivo.Image cited from Yang et al. (2024).
Furthermore,  Guan H et al. (2023)  combined IONPs, Piezo1 activators and zoledronic acid in a hybrid system for the treatment of OP, which was shown to have an excellent therapeutic effect.Also, Yuanyuan Guo et al. (2021) used the IONPs to remote-controllable deliver estradiol to treat OP in rats models and achieved a success.However, the potential mechanism by which IONPs regulate OP is still unclear.In our previous work, we found that IONPs could regulate the inflammatory microenvironment in bone tissues, www.ecmjournal.orgEuropeanCells and Materials Vol.48 2024 (pages 45-65) DOI: 10.22203/eCM.v048a04

Table 1 . The comparison of different synthesis method of iron oxide nanoparticles (IONPs). Method Complexity Energy intensity Homogeneity Shape control Crystallinity Yield
EuropeanCells and Materials Vol.48 2024 (pages 45-65) DOI: 10.22203/eCM.v048a04 Table 1 for the future reference.The more www.ecmjournal.org

Table 3 . The researches of the effect of IONPs on osteogenic differentiation.
(Liu et al., 2020)