Nanotechnological Strategies for Osteoarthritis Diagnosis, Monitoring, Clinical Management, and Regenerative Medicine: Recent Advances and Future Opportunities

Purpose of Review In this review article, we discuss the potential for employing nanotechnological strategies for the diagnosis, monitoring, and clinical management of osteoarthritis (OA) and explore how nanotechnology is being integrated rapidly into regenerative medicine for OA and related osteoarticular disorders. Recent Findings We review recent advances in this rapidly emerging field and discuss future opportunities for innovations in enhanced diagnosis, prognosis, and treatment of OA and other osteoarticular disorders, the smart delivery of drugs and biological agents, and the development of biomimetic regenerative platforms to support cell and gene therapies for arresting OA and promoting cartilage and bone repair. Summary Nanotubes, magnetic nanoparticles, and other nanotechnology-based drug and gene delivery systems may be used for targeting molecular pathways and pathogenic mechanisms involved in OA development. Nanocomposites are also being explored as potential tools for promoting cartilage repair. Nanotechnology platforms may be combined with cell, gene, and biological therapies for the development of a new generation of future OA therapeutics. Graphical Abstract


Introduction
Significant progress has been made in recent years in nanotechnology and nanomedicine. Nanotechnologies are used to deliver anticancer therapeutics, to perform minimally invasive image-guided delivery of plasmids and non-coding RNAs [1], and to facilitate the targeted delivery of conventional and biological drugs [2]. The main benefit of employing nanocarriers in the therapeutics arena is to achieve targeted delivery using the optimum drug dosage, extend drug circulation, reduce side effects, and decrease the likelihood of developing drug resistance. Nanotechnologies provide new platforms for achieving sustained drug release, preventing "burst release" and countering drug resistance.
Currently, nanoparticles (NPs) are the most innovative biomaterials for potential diagnosis and management of osteoarthritis (OA) [3][4][5][6]. Nanomaterials such as liposomes, micelles, carbon nanoallotropes, and quantum dots are described as particles with sizes in the range of 1-100 nm [7,8]. One of the important benefits of nanomedicine is the capability to design special NPs for detection of early osteoarthritic changes in cartilage tissue, e.g., using a liposome containing an antibody to type II collagen, which when combined with a dye emitting near-infrared light enables detection with This article is part of the Topical Collection on Osteoarthritis Highlights • Nanotechnology platforms have potential for implementation in the diagnosis, monitoring, and clinical management of osteoarthritis. • Functional nanomaterials and other nanotechnology-based drug and gene delivery systems may be used for targeting molecular pathways and pathogenic mechanisms involved in osteoarthritis development. • Integration of nanocomposites with regenerative medicine approaches is a strategy for biomimetic cartilage repair. • In the future, nanotechnology platforms may be combined with cell, gene, and biological therapies for the development of a new generation of innovative osteoarthritis therapeutics.
in vivo optical imaging techniques [9]. Furthermore, NPs containing anti-inflammatory drugs and proteins (i.e., anabolic growth factors) are able to release these therapeutics in a prolonged fashion, ensuring sustained release and delivery, which is an important goal for disease therapy [10,11]. However, the side effects of these drugs increase with higher doses. These drugs can be loaded on nanocarriers to reduce and optimize dosage and mitigate their side effects. A variety of bio-based materials such as chitosan, bovine serum albumin, hyaluronic acid (HA), and chondroitin sulfate can be used for the synthesis of NPs [12][13][14][15][16][17][18][19][20]. Liposomes are extensively used for drug delivery in OA due to their biodegradability, biocompatibility, and high encapsulation capacity, as well as the ability to entrap hydrophilic and lipophilic drugs [21]. This approach has been applied for intra-articular delivery of several non-steroidal anti-inflammatory drugs (NSAIDs) to prevent gastric ulceration and other side effects.
Micelles are beneficial in delivery of siRNA [22]. Quantum dots [8] are effective for the recognition of MMP activity in damaged cartilage and other tissues, particularly those coated with streptavidin and conjugated with biotinylated peptide ligands [23]. The aim of this narrative review is to highlight opportunities for the application of nanotechnologies in OA diagnostics, treatment, and regenerative therapy of articular tissues. We propose that nanotechnologies may offer new opportunities and advantages for the diagnosis, prognostic indication, and treatment of osteoarticular disorders, the smart delivery of novel and conventional drugs and biological agents, and the development of biomimetic regenerative platforms for delivering gene and cell therapies to promote cartilage and bone repair.

Osteoarthritis: From Incidence to Clinical Management
OA is the most common form of degenerative joint disease and one of the most chronic musculoskeletal diseases affecting 240 million people across the world [24][25][26][27][28][29][30]. In the USA alone, the cost of treatment is just over $185 billion per year. The impact of OA on society is substantial, grossly underestimated, and increasingly a cause of concern about the ability of healthcare systems to cope with the rising socioeconomic burden.
OA ordinarily manifests in knees, hips, hands, spine, and to a lesser extent in ankles and feet (Fig. 1). The most important risk factors for the development of OA include age, overweight/obesity, joint trauma/instability, gender, genetics, and metabolic/endocrine diseases such as diabetes and crystal deposition disorders such as gout [29] (Fig. 2). Low-grade inflammation [31][32][33] and abnormal mechanical load [34][35][36] are important contributors to the onset and progression of OA [37], leading to the impaired balance between anabolic and catabolic activities in the joint [38]. Genetic factors are associated with OA, 39 to 65% for knee OA and up to 60% for hip OA [39,40]. Since OA is an age-related disease, its incidence is higher in people between 55 and 64 years [41]. Gender is an important risk factor in the pathogenesis of OA. The prevalence, incidence, location, and severity of OA are different in men and women. Although overall the incidence rate of OA is higher in males, as compared to females [42], estimates of World Health Organization (WHO) suggest that the incidence of OA in men and women older than 60 years of age is 9.6% and 18%, respectively [43].
Although increased human life expectancy is associated with increased incidence of OA, there has been an alarming rise in the incidence of OA since the beginning of the postindustrial era. A study conducted by Wallace et al. [44] has suggested that post-industrial modernization is associated with the higher incidence of OA, such that the incidence of knee OA has been higher than early industrial and prehistoric eras. Moreover, they demonstrated that enhancements in longevity and body mass index (BMI) have not been the major reasons for the prevalence of knee OA in the USA since the mid-twentieth century.
In contrast to rheumatoid arthritis (RA), for which many treatments are currently available, there is no effective treatment for OA. A number of pharmacological and nonpharmacological therapies have been developed for the management of OA, which are largely based on symptom modification, decreasing pain, and increasing joint performance [45]. However, disease modification in OA has remained a major challenge, and the futility of currently available treatment is a source of frustration for OA patients and healthcare professionals. NSAIDs are usually administered for symptom modification. However, NSAIDs are not disease-modifying agents and, therefore, do not alter the course of disease progression. Furthermore, their long-term administration is associated with adverse side effects on the renal, gastrointestinal, and cardiovascular systems [46]. In order to remedy OA, there are currently two options in clinical use: (1) nonpharmacological treatments and (2) pharmacological treatments (Fig. 2). For non-pharmacological therapy, a number of guidelines are available along with recommendations that may be presented to the patients, including weight loss, aerobic exercise, and self-management [47][48][49][50]. Metabolic syndrome [51,52] and immunometabolic alterations [53,54], obesity [55], dyslipidemia [56], hyperglycemia, and insulin resistance [57] are all associated with the increased rate of OA. Therefore, physical exercise and weight loss are considered as the most effective interventions for the prevention and treatment of OA [47,48]. With regard to pharmacological therapies that are currently in clinical use, they not only address the symptoms of the disease (i.e., pain), but also aim to impact on the progression of the disease. In line with this strategy, a variety of nutraceutical supplements such as diacerein, glucosamine, and chondroitin sulfate have been examined [58]. Biological agents that target cartilage extracellular matrix (ECM) degradation, bone remodeling, inflammation, and dysfunction of skeletal muscle, as well as adipose tissue metabolism, are considered to be potential candidates for the treatment of OA. Although these approaches seem promising, there is still no effective, approved treatment for OA, which could eliminate the necessity of surgical intervention in most of the cases. Arthroscopic debridement, allograft application, autologous chondrocyte implantation, and matrix-based autologous chondrocyte implantation are methods commonly used to repair focal and isolated cartilage lesions [59][60][61]. These methods allow partial restoration of mobility and help to ameliorate the symptoms of cartilage damage [62]. However, the newly formed tissue, which primarily consists of fibrocartilage, a "scar" tissue, instead of native hyaline cartilage, is very fragile and mechanically weak. Therefore, tissue engineering techniques applying three-dimensional (3D) scaffolds loaded with cells, as well as nanocompound-based drug delivery systems, seem promising approaches for the development of new therapies for cartilage lesions [63,64]. Molecular components and biochemical signals that control differentiated chondrocyte function and promote proper cartilage structure formation, pore size, and mechanical competence should be carefully considered when developing such techniques for the regeneration of damaged cartilage tissue [65]. However, total joint replacement surgery, such as total knee or hip replacement, has been demonstrated as the most effective last-resort treatment for severe forms of OA [66,67]. Therefore, it is vital to develop novel tools and strategies for early diagnosis and treatment of OA.

Novel Pharmacological Treatments and Molecular Pathways
In recent years, much attention has been focused on the elucidation of the molecular pathways involved in the onset and progression of OA, and autophagy is one of the most interesting and promising areas. Research suggests that the loss of autophagy, a homeostatic mechanism, is related to the pathogenesis of OA, as reduced autophagy can be observed in cartilage in late OA consistent with increased levels of chondrocyte apoptosis [68,  69]. A large body of evidence is emerging to suggest a role for peroxisome proliferator-activated receptor gamma (PPARγ) in preserving mTOR signaling, which leads to the inhibition of autophagy [70]. On the other hand, findings have demonstrated the dual role of transforming growth factorbeta (TGF-β) signaling pathways in articular cartilage homeostasis via inhibition of terminal chondrocyte maturation and in the pathogenesis of OA by induction of pathological alterations in the subchondral bone [14,71]. Thus, the regulation of this important signaling pathway can be considered among the potential strategies for inhibiting OA progression. Other pathways such as heparin-binding epidermal growth factor-like growth factor (HB-EGF) [72], fibroblast growth factor (FGF) [73], and hypoxia-inducible factor 1α [74] are under investigation to identify targets to prevent and treat OA.
According to various published guidelines for management of OA, the pharmacologic option is a priority after exercise, self-management, and education. Among pharmacological approaches, natural products have attracted much attention due to their valuable biological properties and low probability of side effects [75,76]. Chondroitin, avocado/ soybean unsaponifiables, curcumin, methylsulfonylmethane, willow bark extract, and pycnogenol are the most popular natural products for modulation and management of OA. However, the discussion of nutrition, dietary factors, and nutritional supplements containing natural products is beyond the scope of this review, and therefore, we would like to direct readers to several recent narrative and systematic reviews on this topic [77][78][79][80][81].

Nanoparticles for Imaging Diagnostics
Although radiography is still the first and most widely used imaging method for assessment of a patient with a suspected or known diagnosis of OA, precise measurement of articular structures is not possible by X-ray. Computed tomography (CT), which is another radiogrphic technique, can also only indirectly assess cartilage degeneration by 3D observation of joint space narrowing, which is a feature of late-stage OA [82,83]. While it is possible to use a contrast agent in CT to directly visualize articular cartilage in 3D in vivo [84], this approach is invasive as it requires an injection of the contrast agent directly into the joint space.
More sensitive non-invasive imagining techniques, for example, ultrasound and magnetic resonance imaging (MRI), allow visualization of changes in the cartilage volume or thickness. In clinical medicine, the most commonly used noninvasive medical imaging technique to visualize the structural changes associated with functional changes in tissues is MRI [85][86][87]. Moreover, by developing new contrast agents, MRI can be applied to permit the more accurate visualization of structural tissue changes. For example, due to high biocompatibility and low toxicity, iron-based magnetic nanoparticles (MNPs) have been developed for clinical oncology imaging as novel biomarker-specific agents [88]. Furthermore, MNPs could also be used as multifunctional agents because it is possible to combine diagnostic and therapeutic properties into them [89]. For example, superparamagnetic iron oxide nanoparticles (SPIONs) exhibit a high degree of saturation magnetization, which is lost in the absence of magnetic field. These NPs are considered relatively less toxic than optical agents. For active targeting, SPIONs can be embedded into polymer cores and conjugated with different peptides, antibodies, or small molecules. In this way, modified SPIONs could serve as agents for the detection of OA biomarkers in synovial fluid [90].
Iron oxide NPs have also been proposed for use in apoptosis detection in transplanted stem cells in arthritic joints. A significant problem for the long-term success of the matrixassociated stem cell implants (MASI) or chondrocyte implants (MACI) is the loss of cells after implantation due to cell migration, necrosis, and apoptosis [91,92]. Clinically, the success of implantation is usually assessed only a few weeks following the cell implantation by invasive arthroscopy and biopsy [93][94][95]. Therefore, early detection and visualization of cell implants is an essential aspect in the development of similar strategies for OA treatments. In vitro studies have shown that iron oxide particles directly accumulates in the cytoplasm of viable cells, while ferumoxides are dispersed in large amounts in smaller cellular fragments following apoptotis. In addition, dispersed iron oxides showed a stronger T2 signal in apoptotic cell fragments compared to iron oxide clusters in viable cells [96].
Non-iron-based NPs, for instance, gadolinium (Gd)-based contrast agent and caspase-3-sensitive nano aggregation MRI probe (C-SNAM), may also be successfully used for MACI and MASI implantation assessment. C-SNAM is a small molecular probe that can be easily delivered to MASI in cartilage defects by injection and passive diffusion. Induction-spectral plasma (ICP-MS) analysis showed significantly higher levels of Gd concentrations in apoptotic adipose-derived stem cells (ASCs) than in viable ASCs. Moreover bioluminescence imaging studies confirmed apoptosis of mitomycin C-exposed cells. In vivo studies demonstrated that strong bioluminescent signals were detected in all ASCs implants immediately after implantation. In addition, C-SNAM-exposed apoptotic cells also showed significantly stronger T1 signals on MRI than viable cells [97]. However, it should be noted that many visualization methods are still in the experimental phase and are not yet standardized sufficiently for use in classification during daily clinical practice [97]. However, it should be noted that many visualization methods are still in the experimental phase and are not yet standardized sufficiently for use in classification during daily clinical practice [98].
A novel and innovative alternative to MRI for detection of cartilage degeneration is photoacoustic imaging (PAI). PAI is a hybrid bioimaging technology that combines the benefits of ultrasound with deep penetration into tissues and optical imaging with high spatial resolution [99,100]. Although the optical image has limited use for detection of OA, PAI can visualize neovascularity in arthritic joints, as well as provide morphological information on the degeneration of finger joints [101,102]. However, contrast agents used currently for PAI lack the sensitivity and specificity for detecting cartilage lesions in the early stages of the disease. NP-based contrast agents such as cationic poly-L-lysine-enveloped watersoluble anionic melanin nanoparticles (PLL-MNPs), which have desirable properties of biocompatibility and low cytotoxicity, could be used to improve the sensitivity of PAI [103][104][105]. Research shows that PLL-MNPs can enhance PAI with significantly different accumulation in OA compared to healthy joints [106]. Interestingly, comparative analysis of results from PAI, radiography, and MRI show that PAI based on PLL-MNPs could be more useful for the detection of cartilage degeneration than standard joint examination methods. Moreover, histological results are consistent with PAI and confirmed the feasibility of PAI using PLL-MNPs to detect cartilage degeneration in early-stage disease [106].
One of the obstacles in evaluating pharmacological intervention involves the definition, identification, and quantification of early OA, as well as following up the efficacy of the applied therapies. Therefore, early detection and accurate visualization of cartilage degeneration and other synovial joint alterations are crucial for the appropriate treatment of OA.

Nanomaterial-Based Biosensors and Biomarkers for Early Diagnosis
The current clinical diagnosis of OA is based traditionally on clinical symptoms (e.g., pain and loss of function) and radiographic criteria (e.g., joint space width), which often occur late in the disease course. An attractive and practical alternative could be the measurement of biochemical markers, which can reflect dynamic events such ECM synthesis and degradation. Biomarkers may be measured in body fluids such as synovial fluid, blood (serum or plasma), and urine [107]. The discovery of a definitive biomarker and its utilization in clinical practice could help to diagnose the disease much earlier and, importantly, to distinguish between phenotypes [108], the fast and slow progressive forms of the disease, and the erosive and non-erosive forms of OA [109,110]. For these reasons, the main proteins of the ECM of cartilage, the metabolic products, and inflammatory mediators are widely examined. Many of these biomarkers are associated with the metabolism of collagen type II or aggrecan in cartilage or of collagen type I in subchondral bone [111][112][113].
Other biomarkers that could reflect an initial change of ECM structure are related to a range of non-collagenous matrix proteins, including glycoproteins and proteoglycans, as well as matrix-degrading metalloproteinases. These molecules are constituents of both cartilage and synovium, and they have a role in other metabolic pathways in the joint.
Technological methods that have been developed for OA biomarker detection include enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (RT-PCR), quartz crystal microbalance, mass spectrometry, and electrochemical methods. Some of these methods offer high sensitivity or selectivity; however, they have essential disadvantages not only because they are time-consuming and costly, but also they have reduced precision. For this reason, methods with high sensitivity and selectivity, as well as small sample volume requirements, are under investigation for their potential to detect OA in its early stages. These include nanoparticle-based devices or label-free and real-time biosensors for specific detection of OA biomarkers such as glycosaminoglycans (GAGs) released from degrading cartilage, HA, cytokines, free radicals (NO), and proteinases.
Loss of aggregating proteoglycans and constituent GAGs, which are essential contributors to the structure and biomechanical properties of articular cartilage, usually occurs before significant morphological changes [114].
HA is a linear polysaccharide that is commonly found in synovial fluid, as well as in the skin and other tissues and organs. Its molecular mass ranges from 10 5 to 10 7 Da. This corresponds to 250-25,000 units of disaccharides [115]. High-molecularweight HA (> 1000 kDa) displays immunosuppressive properties [116]. On the other hand, low-molecular-weight HA (typically < 500 kDa) has pro-inflammatory effects and can stimulate the production and secretion of inflammatory cytokines [117]. Therefore, the size distribution and abundance of HA is considered a promising biological indicator of pathophysiology and could be assessed as a target for disease-specific diagnostics [98,118,119].
Currently, to determine the molecular mass of HA or similar molecules, ELISA, size exclusion chromatography (SEC), and multi-angle laser light scattering (MALLS) methods can be used. However, they all have significant limitations, such as limited ability to distinguish molecular weights of molecules (ELISA), limited practical constraints on the number of fractions and samples that can be tested (SEC) or limited accuracy, and are relatively insensitive for low molecular weight fragments (MALLS). For these reasons, a label-free solid-state nanopore sensor was developed using a nanometer-scale aperture formed in a thin membrane as the only fluid connection between two reservoirs of an electrolyte solution [120]. After analyzing the translocation properties of the molecules, the molecular weight of HA or other molecules could be determined on a per molecule basis, and finally, overall size distribution is obtained from only a few hundred events. For example, SSnanopore-based detection method has been tested in the studies of an equine model of OA [120]. Results confirmed that analysis of translocation properties using this method is sufficient for determining the size distribution and physiological concentration of HA in biological fluids and could be a good alternative for assessment of another OA significant molecular biomarkers.
Proteolytic enzymes, such as MMP-3, MMP-13, or ADAMTS, could be other essential biomarkers for the early diagnosis of OA. However, for determination of the activity of MMPs and ADAMTS, anti-neoepitope antibodies that recognize relatively large substrates are usually used. In some cases, new anti-neoepitope antibodies lack specificity for a particular enzyme because other proteases can also produce similar neopeptides [121,122]. Therefore, the use of non-antibody-based methods could be more appropriate for the detection of ADAMTS or other MMPs than current techniques. For instance, a new fluorescent probe (probe ADAMTS-4-D-Au) based on AuNPs to determine the activity of ADAMTS-4 have been developed. The method employs fluorescein isothiocyanate (FITC) linked to the N-terminal of the ADAMTS-4 specific peptide DVQEFRGVTAVIR (Asp-Val-Gln-Glu-Phe-Arg-Gly-Val-Thr-Ala-Val-Ile-Arg) with the FITC-peptide conjugated to AuNPs with a diameter of 7 nm through cysteine by a gold-thiol bond [123]. This probe is stable under physiological conditions, and fluorescence intensity is proportional to the concentration of active ADAMTS-4. When this ADAMTS-4-D-Au probe was used to determine ADAMTS-4 activity in human synovial fluid, the strongest fluorescence signals were detected in patients with acute joint injury and patients with late-stage OA [123].
Moreover, the high activity of ADAMTS-4 was consistent with the results obtained from the arthroscopy analysis, while the MRI results were different. For instance, for a patient from the group with acute joint damage, arthroscopy revealed second-degree cartilage damage; however, T1-weighted and T2-weighted MRI did not specify any difference related to cartilage damage, while fluorescence intensity in this patient was comparatively high. This probe can be used to identify ADAMTS as a potential biomarker associated with cartilage damage at an early stage of the disease [123].
Alternative non-antibody-based method for non-invasive, real-time evaluation of OA has been developed for the monitoring of the nitric oxide (NO) release in OA chondrocytes, which overexpress the gene encoding inducible NO synthase (NOS2) and its product, NO [124,125]. NO has been considered as a biomarker for OA [126].  [127].
Another promising technology for OA diagnostics with improved sensitivity and analysis time is based on the chemical properties of gold nanoparticles (AuNPs), which are being extensively studied for the development of new multimodal contrast elements or biosensors. Gold nanoparticle biosensing involves the interaction between a target biomarker molecule and a AuNP crosslinker or a AuNP-containing antibody [128].
For biosensor applications, AuNPs are attractive for their chemical stability and convenient spectral window in the visible range. The AuNP-based biosensors are designed to be selective for the detection of various biomolecules, including small molecules, peptides, and nucleic acids.
Particle plasmon resonance (PPR) or localized surface plasmon resonance (LSPR) methods are used widely for the determination of chemical and biochemical species, because of the sensitivity of their electron-rich surfaces to the surrounding environment [129][130][131]. For example, a fiber-optic particle plasmon resonance (FOPPR)-sensing platform, based on AuNP-modified optical fiber for the detection of the OA related pro-inflammatory cytokine interleukin (IL)-1β in synovial fluid samples, has been developed [132]. In this FOPPR sensing platform, the molecular binding of IL-1β on the AuNP-conjugated anti-IL-1β transduces a local increase in the refractive index of the medium surrounding the AuNP, enhancing the plasmon absorbance of the AuNP. Linear regression analysis showed a good correlation coefficient for both ELISA and modified FOPPR detection methods. However, using a FOPPR sensor, the analysis time for detecting IL-1β in synovial fluid was significantly shorter than with ELISA, thereby reducing the chance of potential experimental errors. Similar results were obtained using FOPPR sensor for detection of TNF-α and MMP-3, achieving an excellent refractive index resolution (5.18 × 10-7 RIU) with limits of detection as low as 0.48 pM and 1.56 pM for TNF-α and MMP-3, respectively. This suggests that the label-free and real-time detection capabilities of the FOPPR sensor for protein analysis could be an excellent alternative to immunoassay [133].

Nanoparticles for Drug Delivery
Studies have shown beneficial properties of NPs in systems for targeted drug delivery and sustained release, making them attractive tools for OA treatment ( Table 1). Investigation of selfassembled thermoresponsive nanostructures of HA conjugates [156], administered through subcutaneous and intra-articular injections, has shown beneficial features, including good biocompatibility, sustained drug release, cartilage protection, reduction of inflammatory cytokines such as IL-1β and TNF-α, and maintenance of epiphysis thickness. Nanocrystal-polymer particles have been designed as potential drug delivery carriers for OA treatment [154]. Nanocrystals (NPPs) of kartogenin (KGN), prepared by wet milling and loaded subsequently with polymer microparticles (320 nm), demonstrated high drug loading (31.5% w/w) and prolonged drug release (62% over 3 months).
In vitro experiments showed that KGN-NPPs do not change the mitochondrial activity of cultured human OA synoviocytes. In a murine mechanistic OA model in vivo, the KGN-NPPs show higher bioactivity compared to KGN in solution and sustained intra-articular persistence without any irritation [154]. On the other hand, p38 MAPK inhibitor (PH-797804)-loaded nanostructures (PH-NPPs) are promising for the management of OA [155]. The PH nanocrystals prepared by wet milling and embedded into fluorescent particles were stabilized with D-ɑ-tocopheryl polyethylene glycol 1000 succinate. The PH-NPPs showed beneficial properties in terms of good diameter (14.2 μm), high drug loading (31.5%), prolonged drug release (20% PH release within 3 months), and biocompatibility; PH-NPPs resided in the joint and adjacent tissues for 2 months, associated with decreased levels of inflammatory factors, IL-1β, IL-6, and IL-17, and attenuated inflammation and joint damage.
Furthermore, Avidin nanocarriers are appropriate for intracartilage delivery of dexamethasone (DEX) [159]. Using DEX-Avidin conjugates, prepared using fast (ester) and slow, pHsensitive release (hydrazine) linkers, the DEX was rapidly released from the conjugates, resulting in high bioactivity. In cartilage explants in vitro, a single dose of Avidin-DEX significantly inhibits the cytokine-induced loss of sulfated-glycosaminoglycan (sGAG), as well as decreasing and even suppressing IL-1αinduced cell death and enhancing sGAG synthesis levels [159]. As a novel strategy in the treatment of OA, KGN-conjugated chitosan nano-microparticles can promote cartilage regeneration [15]. These carriers demonstrate excellent properties in terms of prolonged release (7 weeks), strong stimulatory effects on the expression of chondrogenic markers in vitro, long retention time in the knee joint after intra-articular injection, and inhibitory effects on cartilage degeneration in vivo. KGN-conjugated polyurethane NPs (PN-KGN) have demonstrated great potential for OA treatment [160]. These spherical nanocarriers with a mean size of 25 nm release KGN in a sustained behavior. Notably, the nanocarriers are biocompatible, having no cytotoxicity or proinflammatory impact on cells. Intra-articular administration of these NPs decreases cartilage degeneration remarkably, resulting in inhibition of OA development [160]. Polypeptide nanogels (PNGs) with encapsulated methotrexate (MTX) were investigated for improving collagen-induced arthritis [161]. PNG-MTX showed glutathione (GSH)-triggered release behavior similar to that of MTX alone. Moreover, PNG-MTX had high internalization and toxicity against activated macrophages. PNG-MTX treatment in vivo remarkably decreases arthritic scores and diminishes paw thickness, suggesting potential anti-inflammatory activity. Histopathological analysis of the PNG-MTX group revealed decreased numbers of inflammatory cells, normalization of cartilage morphology, joint space widening, and decreased roughening of the articular surface [161].
Silk fibroin microparticles (SFMs) have been designed for intra-articular drug delivery [162]. SFMs are spherical in shape with particle size in the range of 598 nm to 21.5 μm and show prolonged release and retention in the joint. Furthermore, curcumin-loaded solid lipid nanoparticles (Cur-SLNs) exhibit high efficacy for alleviation of adjuvantinduced arthritis [163]. Cur-SLNs (10 and 30 mg/kg) effectively decreased joint hyperalgesia, joint stiffness, and paw volume, as well as improving the mobility score, reducing blood leukocyte count, and decreasing oxidative stress, TNF-α, and C-reactive protein. HA-chitosan nanoparticles (HA-CNPs) are also appropriate for delivery of curcuminoid in knee OA treatment because of their high drug loading capacity (38.44%) and prolonged drug release behavior [164]. In a knee OA model, using the Hulth-Telhag surgical procedure and co-treatment with IL-1β and TNF-α, the administration of curcuminoid-loaded HA-CNPs significantly reduced the Outerbridge and Mankin pathological scores to close to normal until the fourth week. The curcuminoidloaded HA-CNPs also significantly suppressed NF-kB signaling and expression of the metalloproteinases MMP-1 and MMP-13, whereas they upregulated the collagen II expression in chondrocytes in vitro [164].

Nanoparticles for Gene Delivery and Gene Therapy
The challenges of chemoresistance to drugs and associated side effects have opened up new opportunities in the field of gene therapy [165], offering novel perspectives for the design of biocompatible, biomimetic, and efficient gene carriers.
For instance, carbon dots in complex with the gene encoding TNF-α have been designed to facilitate stem cell-based PVCL-co-acrylic hydrogels Sodium diclofenac -Sustained permeation through an artificial skin membrane and high drug delivery [138] Lipid nanoparticle Ibuprofen Male SKH-1 hairless mice High entrapment efficiency (95.51%), high permeation, and potential anti-inflammatory activity [139] PLGA nanoparticle IL-1 receptor antagonist (IL-1Ra)

NF-KB inducible reporter cell line
Tunable size (300-700 nm), cytocompatible, good stability, and efficient inhibition of IL-1β signaling [140] AuNPs Chondroitin sulfate Primary goat chondrocytes High increase in GAG and collagen production, stimulating chondrocyte proliferation, and enhancing extracellular matrix production [141] Chitosan NPs Berberine Rat knee OA model Spherical shape, good stability, ideal releasing profile, increased retention time in synovial fluid and high anti-apoptotic activity [20] Solid lipid NPs Aceclofenac Albino rat Good particle size (143.4-154.2 nm), prolonged drug release, high uptake, and great anti-inflammatory activity [12] Lipid NPs Diacerein Rat model of OA Good particle size (396 nm), sustained release, high delivery, and improved histopathology analysis [142] Lipid NPs Diacerein Rat Good particle size (270 nm) and zeta potential (− 13.78 to − 19.66 mV), high entrapment efficiency (88.1%), sustained drug release, and decreased side effects of diacerein [143] NPs-in-microspheres Brucine Rats High biocompatibility, prolonged drug release, high residence in articular cavity, and improved retention [14] Polymeric NPs KAFAK THP-1 cells Cartilage plugs Decreased pro-inflammatory cytokine and selective targeting [144] Polymeric NPs Curcumin Human primary chondrocytes Inhibiting mRNA expression of pro-inflammatory mediators (IL-1β; TNF-α; MMPs 1, 3, and 13), decreasing OA disease progression, reducing proteoglycan loss, and decreasing synovitis [145] PEGylated NPs KAFAK Chondrocytes Efficient targeted delivery and decreasing inflammatory reaction [146] Polymeric NPs IL-1Ra protein Synoviocytes Rat stifle joint Good particle size (300 nm), maintaining bioactivity, specifically targeting synoviocytes, increased retention time, and decreasing inflammatory factors [19] Bipolymeric NPs Dextran FITC Healthy rat knees No decrease in proteoglycans biosynthesis and induction no inflammatory response [147] PLGA NPs Dexamethasone Synovium and articular damage Excellent biocompatibility, internalization via phagocyte process and stimulation of inflammation [148] Polymeric NPs Curcumin Rat model of OA Enhancing cellularity and matrix and high biocompatibility [149] Coiled-coil protein BMS493 Human articular chondrocytes Reducing mRNA levels of MMP-13 and IL-1β [150] Ginger extract nanoparticle -Patients with knee OA Improving knee joint pain, symptoms, daily activities, and quality of life [151] Solid lipid NPs Diacerein -High encapsulation of diacerein, prolonged release behavior, increase in diacerein payload and thermoresponsive drug delivery [152] Amine terminal polyamidoamine (PAMAM) dendrimres Insulin-like growth factor 1 (IGF-1) Rat OA Promoting pharmacokinetics and potential of disease-modifying OA drugs [153] Nanocrystals-polymer particles -Human OA synoviocytes and murine mechanistic OA model Lack of effect on mitochondrial activity, exerting protective effect on the cartilage and epiphysis of the medial tibia, and significant reduction in VEGF and Adamts5 expression [154] Nanocrystal-polymer particles P38a/b MAPK inhibitor PH-797804

OA model and human OA synoviocytes
Lack of toxicity against human OA synoviocytes, decreasing inflammation and joint destruction and also excellent retention and function at the target site [155] Self-assembled thermoresponsive nanostructures of hyaluronic acid OA mouse model High biocompatibility and significant sustained residence time at the injection site, reduction of inflammatory cytokines and efficacy in delivery of peptides, proteins or small molecules [156] Poly (ester-amide) particle Celecoxib Ovine model High biocompatibility, no toxic effect at the injection site, and great diffusion into neighbor tissues [157] HA-PLGA particles -

RAW264.7 macrophage cells and Wistar rats
Great safety and high anti-inflammatory effect [158] therapy of cartilage defects [179]. In a rabbit OA model, chitosan-graft-polyethylenimine (PEI)-DNA NPs [170], which demonstrate high transfection efficiency and good biocompatibility confirmed by cell viability assay, is able to deliver plasmid-DNA (pDNA) into the nuclei of chondrocytes and synoviocytes. Image-guiding, photothermal-triggered hemoglobin (Hb)-based NPs can absorb near-infrared light at 650 nm (0.5 W cm −2 ) and convert it into heat. The Notch1-siRNA-loaded NPs act by suppressing macrophage inflammation, suggesting that this is potential biocompatible nano-platform for clinical OA therapy [179]. HA-chitosan-NPs [172] transfect chondrocytes with high efficiency and maintain cell viability at more than 90% [172]. Chitosan NPs carrying the gene encoding IL-1Ra exhibit high efficacy for gene delivery [173], and when injected into the knee joints of rabbits with OA, increase the levels of IL-1Ra in knee joint synovial fluid. In contrast, no IL-1Ra can be detected in the chitosan-IL-10-injected group. In addition, decreased severity of histologic cartilage lesions was detected in the group treated with chitosan-IL-1Ra [173]. Chitosan-HA-NPs carrying pDNA encoding IL-1Ra exhibit advantageous effects in alleviation of inflammation in synoviocytes, where they enhance IL-1Ra gene expression and decrease the mRNA and protein levels of MMP-3, MMP-13, cyclooxygenase-2 (COX-2), IL-1β, and iNOS [174]. These nanocarriers have zeta potential and particle size of + 28 mV and 144.9 nm, respectively, and effectively protect pDNA. The pDNA exhibits a prolonged release pattern of up to 15 days, and the biocompatibility of these nanocarriers can be confirmed by cytotoxicity assay [174].
As described above, alginate can be easily used not only as a scaffold for cell culture, but also as a nanocarrier for delivering genes to cells. Gene-activated alginate hydrogels capable of non-viral gene delivery via nanohydroxyapatite (nHP) have been developed to control differentiation potential of mesenchymal stem cells (MSCs) for either cartilage or endochondral bone tissue engineering [175]. For this purpose, MSCs and nHP complexed with DNA, encoding TGF-β3, BMP-2, or a combination of both (TGF-β3/ BMP2), were encapsulated into alginate hydrogels [175].
Nanotubes comprise another interesting tool applied in cartilage tissue engineering, since they can be delivered directly to the cytoplasm in the cell. Among the different types of nanotubes, polyethylene glycol (PEG) chain-modified single-walled carbon nanotubes (PEG-SWCNTs) were able to efficiently enter the cartilage ECM, translocate into the cytoplasm of chondrocytes, and deliver gene inhibitors without affecting cartilage homeostasis [180]. This approach can control molecular functions of cells, which is an important option for improving cellular differentiation capability. Arthritic rats Biocompatible, improved siRNA stability, high uptake by macrophages, and great anti-inflammatory effect [169] Chitosan NPs DNA (plasmid) Chondrocytes and synoviocytes High transfection efficiency, great biocompatibility, and delivery of pDNA into the nucleus of chondrocytes and synoviocytes [170] Calcium phosphate/liposome NPs NF-kB targeted DNA Arthritic rats Inhibiting the progression of OA by targeting macrophages and decreasing pro-inflammatory cytokines by inhibiting NF-kB signaling pathway [171] Hyaluronic acid/chitosan NPs Plasmid-DNA Chondrocytes High transfection efficiency and increasing the viability of chondrocytes [172] Chitosan NPs IL-1Ra or IL-10 genes Osteoarthritic rabbits Improving histologic lesions and decreasing inflammation [173] Chitosan-HA NPs IL-1Ra Synoviocytes Sustained pDNA release, high biocompatibility, and great anti-inflammatory effect [174] Nanohydroxyapatite (nHA) TGF-β3 and BMP2 MSCs Directing MSCs fate for articular cartilage and endochondral bone tissue engineering [175] Polymeric NPs Anti-Hif-2α siRNA Arthritic mice Downregulation of Hif-2α, MMP-12 and -9, ADAMTS-4, VEGF, collagen type X and NF-kB, promoting local concentration, increasing retention time, decreasing IL-1β and attenuation of synovium inflammation [176] HA/chitosan NPs Cytokine response modifier A Rat knee osteoarthritis model Effective entrapment of plasmid-DNA, sustained release over 3 weeks, inhibiting cartilage damage, synovial inflammation, and loss of type II collagen and downregulation of IL-1β and MMP-3 and MMP-13 [177] Bioconjugated carbon dots with succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) Silenced TNF-α (siTnfα) MSCs MSCs chondrogenesis enhancement by inflammation suppression [178] NO-hemoglobin@PLGA-PEG NPs Notch1-siRNA Macrophage Suppressing macrophage inflammation [179] Nanomaterials-Based Scaffolds for Cartilage Regeneration Scaffolds are a key element that enables tissue regeneration. The requirements to create a scaffold include biocompatibility; complex structure (hierarchy and porosity); mechanical strength and flexibility; promotion of cell attachment, migration, and proliferation; and minimal inflammatory and immunological response. In addition to hierarchical structure and porosity of scaffolds, nano-topography is an important cue for cell adhesion, proliferation, and differentiation [181]. The effect of nano-topography on chondrogenic differentiation of MSCs was investigated via thermal nanoimprinting of PCL film. The results indicated that nano-topographical patterns affect the morphology, cytoskeletal structure, cell aggregation, and differentiation of MSCs, resulting in specific functional outcomes. Furthermore, nanopatterned films enhance chondrogenesis of MSCs and facilitate hyaline cartilage formation compared with smooth films [182].
In addition to electro-spun nanofibers, hybrid peptide nanofiber-HA membrane scaffolds have been developed [191]. This scaffold preserves cartilage morphology, reduces osteophyte formation, and maintains cartilagespecific matrix proteins in OA models in vivo [191]. Self-assembled peptide (SAP) nanofibers coupled with the neuropeptide substance P (SP) [192] has been investigated as an injectable conjugate containing different concentrations of SP and applied in a rat OA model. The results showed that SP-SAP nanofibers can promote chondrogenic differentiation and delay the progression of OA. Following similar strategies, injectable hydrogel scaffolds containing chondroitin sulfate nanoparticles (ChS-NPs) and nanohydroxyapatite (nHA) were developed for osteochondral regeneration and evaluated using a rabbit model [193]. These scaffolds enhanced hyaline cartilage regeneration with subchondral bone formation and lateral host-tissue integration.
Although polymer-based scaffolds have demonstrate good biological compatibility, they quite often lack other important properties such as mechanical strength, failing to deliver the proper cues to promote functional tissue regeneration. To overcome the challenges encountered when using polymerbased scaffolds, researchers have investigated the use of nanomaterials beyond the traditional biomaterials to create nanocomposite scaffolds capable of stimulating cell attachment, growth, and tissue regeneration. Among the available nanomaterials, carbon nanotubes (CNTs) have attracted attention due to their outstanding electrical and mechanical properties, as well as their versatility in assembly of different structures. In the field of tissue engineering in particular, CNTs present nanostructural dimensions in the scale of proteins found in the ECM, enhancing the potential to influence cell attachment, proliferation, and differentiation [194]. Primary chondrocytes proliferate and align on 2D pristine CNT sheets and express high levels of ECM proteins when cultured in 3D pristine CNT textile made out of aligned CNT fibers [195]. CNTs have been also used to reinforce polymer-based scaffolds [194]. Functionalized single-wall CNTs (SWCNTs) can strengthen the mechanical properties of agarose hydrogels, while providing the optimal structure needed to maintain cellular viability and promote cartilaginous growth [196]. Nanocomposite films for chondrocyte growth have also been created using highly dispersed CNTs in polycarbonate urethane (PCU) [197], exploiting the possibility to provide electrical stimulation to cells via the conductivity property of CNTs. As a result, chondrocyte attachment and long-term cell densities can be enhanced by more than 50% (without electrical stimulation) and 200% (with electrical stimulation) on CNT-PCU composites compared to pristine PCU.

Conclusions and Future Opportunities
Increasingly rapid development of nanotechnologies has offered a wide variety of novel approaches and platforms for both diagnosis and regenerative treatment in OA. Nanotubes, magnetic NPs, and other nanotechnology-based drug and gene delivery systems provide important targeting platforms for the development of OA therapeutic strategies. However, due to the complexity of molecular and cellular alterations in cartilage tissue in OA, nanocomposites are also currently under scrutiny as potential tools for efficiently building cartilage matrix for repair strategies. Many of the current cell-based therapies for OA are relatively simple injections of MSCs, primary chondrocytes, transduced chondrocytes, or mixtures of allogeneic primary chondrocytes and protein production platforms. One relevant example is the Kolon TissueGene cell-based therapy "TissueGene-C (TG-C)." This new and revolutionary cell-based therapy employs GP2-293 cells, a HEK 293-based retroviral packaging cell line used for large-scale growth factor production, in this case transforming growth factor-β1 (TGF-β1). This product concept has the capacity to over-produce TGF-β1 in sufficiently high quantities for supporting cellular therapy and regenerative applications, but the cells are simply injected into the joint, without any supporting gel or matrix. In the future, cell-based therapies will benefit from nanotechnology-based delivery 3D platforms and matrices that can better support the cell-based therapy. Transduced GP2-293 cells in TG-C may be transformed cells, but since they have been irradiated, they have lost their capacity for proliferation and cannot differentiate. After these cells carry out their TGF-β1 production duties, they will die and their remains will be cleared by joint resident inflammatory macrophages through the process of phagocytosis. Therefore, delivering cell-based therapies in a matrix of nanomaterials will enhance their survival and promote macrophage access to the dead cells, allowing the immune system to clear the debris. There is huge potential in this area for new innovations that can promote sustained delivery of chemical and biological drugs and the stabilization of cells for cell-based therapy.
Acknowledgments We would like to thank our collaborators and members of our research teams for useful discussions about the emerging applications of nanotechnology, physical, and spectroscopic technologies in OA diagnosis and treatment.

Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Dedication We dedicate this paper to the memory of our dear colleague Dr. Lauriane Janssen who passed away in November 2019. We will miss her intellect, her infectious enthusiasm, her beautiful smile and her warm personality.