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

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) [3‐6]. 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 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 [12‐20]. 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.

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 [24‐30]. In the USA alone, the cost of treatment is just over $185 billion per year. The impact of OA on society is substantial, grossly under-estimated, 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 [31‐33] and abnormal mechanical load [34‐36] 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 post-industrial 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 non-pharmacological 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) non-pharmacological 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 [47‐50]. 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 [59‐61]. 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.

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 factor-beta (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 [77‐81].

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].

To incorporate nano-topography into free standing scaffolds, electro-spun nanofiber-based scaffolds have been fabricated using different polymers such as poly(ε-caprolactone) (PCL), polyethersulfone (PES), and poly (lactic-co-glycolic acid (PLGA) and evaluated for chondrocyte differentiation [183‐188]. A common finding among these in vitro studies is that nanofiber-based polymer scaffolds enhance chondrogenic differentiation of MSCs. Electro-spun nanofibers have been used in the development of composites with chitosan [189] and collagen [190]. While further studies are needed to evaluate the impact of chitosan in nanofibers-based scaffolds [189], collagen-poly (vinyl alcohol) nanofiber-based scaffolds in a rabbit OA model demonstrates effective regeneration of injured joints [190].

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 cartilage-specific 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 polymer-based 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.

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.