miR-140 is the first miRNA shown to be expressed in cartilage and is among the most abundantly expressed and extensively characterized miRNAs in chondrocytes (17). In the growth plate, miR-140 demonstrates a distinct expression pattern, predominantly in proliferating chondrocytes, as evidenced by RNA in situ hybridization. Its expression is notably minimal in hypertrophic chondrocytes, indicating a potential involvement in early stages of chondrogenic differentiation ( Figure 3A ) (17, 18, 54).

Global deletion of the Mir140 gene leads to a skeletal phenotype characterized by limb shortening, growth plate abnormalities and craniofacial deformities (18, 19, 55). miR-140-null mice display impaired differentiation of resting zone chondrocytes into columnar chondrocytes, while paradoxically accelerating the transition from proliferative to hypertrophic chondrocytes, leading to premature bone formation. However, despite this enhanced hypertrophic progression, the overall disruption in chondrocyte differentiation caused by miR-140 deficiency remains modest (19). Several studies outlined below delve into the broader mechanisms of miR-140 throughout the growth plate.

While BMP signaling promotes the expression of key transcription factors such as SOX9, its activity must be carefully regulated to maintain the balance between proliferation and differentiation within the growth plate (67, 69). In the proliferative zone, BMP activity supports chondrocyte proliferation and extracellular matrix synthesis, while in the prehypertrophic and hypertrophic zones, BMP signaling drives differentiation and hypertrophy (66, 69). For instance, BMP2 enhances the expression of RUNX2 (70), and upregulates COL10A1, a hallmark of hypertrophic differentiation. Indeed, disruption of BMP signaling can result in severe skeletal abnormalities, including shortened long bones and disorganized growth plates, as seen in genetic models with impaired BMP receptor function (66).

miRNAs modulating components of the BMP pathway have the capacity to fine-tune cellular responses to BMP ligands in a zone-specific manner ( Figure 3C ). In one example, an RNA sequencing study comparing cells cultured from resting or prehypertrophic/upper hypertrophic zone of cartilage showed that miR-143-3p was highly expressed in the resting zone chondrocyte cultures (14). In vitro studies in rat mesenchymal cells showed that miR-143-3p targets Bmpr2, which is expressed throughout the growth plate (15, 71). miR-143-3p over expression in vitro decreased cartilage matrix production and decreased cell proliferation, an effect that could be related to its targeting of Ihh (72). Thus, miR-143-3p expression in resting zone cells likely decreases the sensitivity of these cells to BMP ligands, which could help maintain cells in the resting state. Conversely, decreased levels of miR-143-3p in prehypertrophic cells could relieve repression of its target Ihh, allowing expression of this key signaling component ( Figure 2 ).

miR-199a-3p is another miRNA regulating the BMP pathway. miR-199a-3p is decreased during the BMP2-mediated chondrogenic differentiation of C3H10T1/2 cells and it targets Smad1, suggesting that miR-199a-3p is a negative regulator of differentiation ( Figure 3C ) (21). Likewise, miR-1307-3p inhibits the chondrogenic potential of human adipose-derived stromal/stem cells (hADSCs) by targeting BMPR2, emphasizing its inhibitory role in cartilage formation ( Figure 3C ) (73). Together, these miRNAs add a crucial layer of post-transcriptional control to the BMP axis, ensuring its integration into the complex spatial-temporal regulatory networks governing cartilage differentiation.

Other in vitro studies describe miRNAs directly targeting Sox9, notably, miR-495, miR-101, and miR-1247 which act as inhibitors of chondrocyte differentiation ( Figure 3D ) (74–77). Inhibition of miRNAs such as these may be useful for promoting chondrogenic differentiation.

While synthesized primarily by resting zone chondrocytes, PTHrP acts on proliferating zone cells. A miRNA microarray demonstrated a handful of miRNAs regulated by PTHrP treatment during TGFβ-induced chondrogenic differentiation of hMSCs. Of these, miR-892b was modestly induced by PTHrP during the terminal phases of chondrogenic differentiation. Lentiviral-mediated over expression of miR-892b emulated the effects of PTHrP in that it enhanced expression of COL2A1 while inhibiting hypertrophy markers such as COL10A1 (80). miR-892b targets Kruppel-like factor 10 (KLF10/TEIG1), a transcription factor that directly binds to the IHH promoter region to drive transcription, providing potential mechanisms for limiting the premature synthesis of IHH in cells responsive to PTHrP. These investigators also demonstrated miR-892b targeting of Wnt6, a negative regulator of limb development, which may also play a role in regulating hypertrophy.

A robust miRNA profiling study conducted on micro-dissected zones of the rat growth plate identified differential expression of 34 miRNAs between the proliferative and hypertrophic zones (13). Out the 29 highly expressed miRNAs in the proliferative zone, several miRNAs (miR-503-5p, miR-374-5p, miR-379-5p) were prioritized for further study, based on their potential to target a panel of established markers of hypertrophic differentiation ( Figures 4A, B ). The authors hypothesized that expression of these miRNAs could contribute to proliferation, delay hypertrophy and may be induced by PTH1R signaling. Indeed, inhibition of miR-503-5p, miR-374-5p or miR-379-5p in rat primary chondrocytes decreased proliferation and stimulated hypertrophic differentiation specifically affecting Ihh, Bmp2 and Bmp6 expression. Further, these miRNAs were induced by PTH1R signaling in rat growth plate chondrocytes in vitro ( Figure 4B ). The authors proposed a mechanistic model in which the PTHrP concentration gradient across the growth plate zones increases the expression of miR-503-5p, miR-374-5p, and miR-379-5p, which in turn promotes chondrocyte proliferation and inhibits hypertrophic differentiation. This work highlights the interaction between PTHrP signaling and miRNA-mediated gene regulation in growth plate.

The miR-26 family is composed of miR-26a and miR-26b, which are highly conserved across species and have overlapping but distinct roles in regulating key biological pathways, particularly in bone and cartilage development (81).

miR-1 is primarily expressed in the pre-hypertrophic and hypertrophic zones of the growth plate ( Figure 4A ). It has a complex genomic organization, as the Mir1 gene is clustered with Mir133 on two different mouse chromosomes and both Mir1 genes give rise to the same mature miRNA (25). For example, on chromosome 2, Mir1-2 and Mir133a-1 are separated by 9.3 kb and may not be regulated together. In contrast, on chromosome 18, the Mir1-1 and Mir133a-2 genes are separated by 2.5 kb, appear to be within the same pri-miRNA, and both miR-1 and miR-133 are enriched in matrix vesicles (88). miR-1 and miR-133a provide an example of how multiple miRNAs may be co-transcribed within a single pri-miRNA, enabling coordinated expression, although miR-133a targets in the growth plate have not been examined.

As previously discussed, HDAC4 is a negative regulator of chondrocyte hypertrophy in part because it interacts with and inhibits RUNX2 activity, and miR-1 directly targets an evolutionarily conserved region of the Hdac4 3’ UTR ( Figure 4D ) (25). By targeting Hdac4, miR-1 reduces the inhibitory effect of HDAC4 on RUNX2, resulting in increased expression of the RUNX2 target genes Col10a1 and Ihh in vitro (60, 89). A subsequent in vitro study showed direct targeting of Ihh by miR-1 (90), while transgene-mediated overexpression of miR-1 in Col2a1 expressing cells led to shorter limb and body length, delayed formation of secondary ossification centers and delayed terminal differentiation (26). The Col2a1-miR-1 transgenic mice displayed reduced Ihh expression in growth plate chondrocytes both in vivo and in vitro, with decreased expression of key hypertrophic markers such as Runx2, Col10a1, and Mmp13, further impacting chondrocyte hypertrophy. Taken together, these findings suggest that miR-1 is involved in an incoherent regulatory network, modulating both stimulatory and inhibitory factors of hypertrophy ( Figure 4D ).

In mice, the gene coding for miR-322-5p is located within a miRNA cluster on the X chromosome (Mirc24 cluster contains Mir351, Mir503, Mir322) (27). While miR-322 is not found in humans, its human ortholog is miR-424, which is also located on the X-chromosome within a miRNA cluster, along with MIR503. Since work on miR-322/miR-424 function in cartilage has been performed mainly in rodents, our discussion here focuses on miR-322.

miRNA-322-5p is well expressed in the prehypertrophic and hypertrophic zones in mice and during the terminal differentiation of a mouse chondrogenic cell line in vitro ( Figure 4A ). While the majority of miRNAs function as negative regulators of RNA stability and translation, the first studies on miR-322-5p in growth plate showed that this miRNA inhibits the RAF/MEK/ERK pathway in cartilage cells by stabilizing Mek1 RNA. Deletion of the Mirc24 cluster in Col2a1 expressing cells (Col2a1-Cre-Mirc24tm1M) led to shortened hypertrophic zone length, decreased protein levels of SOX9 and SOX6, and corresponding decreases in Acan and Col10a1 levels (27, 91).

A recent in vitro study showed that Sox9 increases the expression of miR-322-5p, which targets Smad7, a negative regulator of TGFβ signaling ( Figure 4E ) (92). The effect of TGFβ signaling in the growth plate is dependent on chondrocyte differentiation stage. For example, deletion of Tgfbr2 in Prx-cre expressing cells results in decreased proliferation, accelerated transition from prehypertrophic to hypertrophic, and delayed late hypertrophic differentiation (93, 94). Since miR-322-5p is expressed in the prehypertrophic and hypertrophic zone cells, it is possible that this miRNA functions to promote hypertrophic differentiation, as it targets a negative regulator of TGFβ signaling (95) ( Figure 4E ).

More translational studies in rats linked miR-322-5p levels in blood exosomes with skeletal size in pups with low birth weight caused by fetal malnutrition (96). Specifically, low birth weight pups that failed to catch up in size after birth had increased levels of miR-322-5p in their liver and in blood exosomes. miR-322-5p levels correlated inversely with body length and growth hormone receptor levels. Subsequently, miR-322-5p was shown to target the growth hormone receptor (GHR), which is expressed in the resting and proliferating zones of the growth plate and mediates the anabolic effects of growth hormone on the growth plate ( Figure 4E ) (97, 98). miR-322-5p is not abundantly expressed in resting and proliferating zone cells, and these studies support the concept that miRNAs from other tissues, such as liver, delivered to the skeleton by exosomes, could have a paracrine function on growth plate activity.

Additionally, research into pregestational diabetes mellitus revealed that maternal hyperglycemia can restrict fetal endochondral bone growth, causing a marked reduction in the length of the hypertrophic zone and decreased miR-322-5p expression in this region (28). Complementary in vitro experiments confirmed that high glucose exposure downregulated miR-322-5p in a chondrogenic cell line, impacting critical markers like Sox9 and Pthlh, which maintain proliferative chondrocytes and delay hypertrophy. Simultaneously, the expression of hypertrophic markers Runx2 and Col10a1 was increased. While this work highlights the potential impact of glucose levels on miRNAs in the growth plate, there is still much to be learned about the miRNA-mRNA target interactions underlying this growth plate pathology. Further, while the study suggests a role for altered miR-322-5p expression in hyperglycemia, the mechanisms by which glucose or metabolic hormones may regulate miR-322 levels remain undetermined.

A recent proof-of-concept study explored the therapeutic potential of targeting miR-338-3p in the treatment of cleidocranial dysplasia (CCD), a genetic bone disorder caused by decreased expression or activity of RUNX2, and characterized by skeletal abnormalities, including short stature and osteopenia, as well as anomalies involving the axial skeleton, skull and teeth (109, 110). Earlier work demonstrated that Runx2 is targeted by miR-338-3p, a miRNA found to be increased in the serum of post-menopausal osteoporosis patients and in mice post-ovariectomy (111). Using mice haploinsufficient for Runx2 as a CCD model, knockout of the Mir338~3065 cluster was able to rescue much of the bone growth defect caused by decreased Runx2 levels, whereas rescue of the skull defects was less prominent (109). Overall, this work suggests that decreasing the expression of miRNAs that target RUNX2 may be a strategy for ameliorating some of the skeletal phenotypes caused by CCD.

Most studies addressing the potential of miRNA-based therapeutics for cartilage disorders have been performed in the context of arthritis, a multifaceted disease impacting articular cartilage and the underlying bone. This is understandable, since the prevalence of arthritis is very high, particularly osteoarthritis, and is associated with significant impacts on quality of life, posing an important economic burden. Moreover, since skeletal stem and progenitor cells are becoming more therapeutically relevant for degenerative musculoskeletal diseases, an understanding of how miRNAs may regulate the persistence of stem-like properties and commitment to specific lineages is of immense interest. In this regard, studies have documented miRNA expression profiles during the in vitro chondrogenic differentiation of mesenchymal stem cells and validated some potential targets of differentially regulated miRNAs. Among the miRNAs reviewed herein, these studies have highlighted functionality for miRNAs including miR-140-5p, miR-143-3p, and miR-199a (21, 112, 113). Another study established a panel of miRNAs that might be useful for determining whether MSCs from human donors may have low or high chondrogenic potential in vitro, which could have an impact on selecting donor cells for use in tissue regeneration applications (114). Fortunately, many of signaling mechanisms and regulatory pathways are highly conserved between articular cartilage and growth plate cartilage (71, 115), so insights into miRNA function in growth plate may be informative for articular cartilage, and some of the more translational work on miRNA-based therapeutics for osteoarthritis may be relevant to the management of growth plate injury.

Much of the work on miRNAs in osteoarthritis has focused on pathways involved in articular cartilage matrix degradation, chondrocyte apoptosis and inflammation. Since miRNAs can act as both promoters and suppressors within these processes, therapeutic potential may be realized through administration of either synthetic miRNA inhibitors (antagomirs; anti-miRs) or mimics (agomirs) (116). Because inflammation, as well as cartilage matrix degradation, is a hallmark of osteoarthritis, miRNAs targeting pro-inflammatory cytokines and signaling molecules are of particular interest. miR-140, for example, is downregulated in human osteoarthritis cartilage samples compared with healthy controls (117). Transfection of chondrocytes with miR-140 mimic antagonized the ability of IL1β to increase expression of the matrix degrading enzyme Adamts5 and the IL1-mediated decrease in Acan (118). Subsequent studies demonstrated that joint destruction was attenuated in transgenic mice expressing miR-140 under the control of the Col2a1 promoter, providing proof-of-concept for a potential miR-140 mimic-based therapeutic. Moreover, targeting of Adamts5 by miR-140 provides a mechanism contributing the protective effect of miR-140 on matrix degradation (18). Extensively studied in both osteoarthritis and growth plate, the role of miR-140 in maintaining both articular cartilage and growth plate function highlights its therapeutic potential. For additional information on miRNA function in osteoarthritis, please consider this excellent review (119).

Beyond osteoarthritis, the potential of miRNA-based therapeutics for addressing cartilage injuries could be explored in an a scenario specifically relevant to the growth plate: the treatment of physeal bony bridge or bony bar formation post-injury in pediatric patients (120). Physeal bars occur most commonly following trauma or fracture. They extend through the growth plate between the epiphysis and metaphysis, resulting in an abnormal bony repair tissue that bridges the two regions. This bony bridge disrupts growth plate function, leading to growth arrest, angular deformities, and limb length discrepancies due to impaired longitudinal bone growth. Thus, it represents a critical issue in pediatric orthopedic care (121).

Physeal injury is fairly common, comprising 15-30% of pediatric fractures. Of these, an estimated that 1-10% of physeal injuries result in bony bridge formation (122). The probability of poor outcome increases with the proportion of the growth plate affected. For example, fractures most likely to demonstrate physeal bar formation are those with fracture through the physis into the epiphysis or those in which the fracture extends from the articular surface to the metaphysis (123). Current treatment primarily involves surgical resection of the bony bridge, with placement of an interpositional material (e.g. cranioplasty cements, cellulose-based matrices, autologous fat graft) to fill the gap. The optimal outcome would be prevention of bony bridge re-formation and regeneration of the native growth plate cartilage, restoring normal bone growth (123). However, the interpositional materials currently in use can either offer mechanical properties but lack the ability to integrate or biodegrade, or those that offer the potential for integration or degradation lack mechanical properties. Moreover, present materials lack the ability to modulate intrinsic pathways to prevent osteogenesis and promote cartilage repair. Because most treatment for bony bridge requires surgical resection and implantation of a material, it provides an avenue for improved, locally delivered therapeutics that may restore growth plate function. Novel therapeutic strategies can be tested for potential efficacy in small animals, such as rats or mice, while larger animals, such as rabbit, mini-pig or sheep are used to test performance in bony bar revision procedures more similar to those performed in patients (123).

The unique cellular environment and regulatory pathways of the growth plate make miRNA-based interventions a potential strategy to minimize adverse outcomes post-injury, by modulating signaling pathways involved in osteogenesis, inflammation, and chondrocyte stability. Further, during fracture repair, chondrocytes can transdifferentiate into osteoblasts; thus an understanding of the mechanisms underlying this process could also be beneficial for designing strategies to restrain bony bar formation (30, 124, 125) Several miRNAs, such as miR-140 and miR-145, are known to regulate chondrocyte differentiation by inhibiting osteogenic signaling, potentially offering a method to prevent abnormal bone formation within the growth plate after injury (19, 126, 127). By selectively targeting pro-osteogenic pathways, localized delivery of these miRNAs might help maintain a chondrogenic microenvironment, preserving the function of the growth plate and preventing bony bridge re-formation. Other miRNAs such as miR-374, miR-379 and miR-503 are crucial for enhancing chondrocyte proliferation; their delivery could foster a pro-regenerative environment by promoting chondrocyte growth and matrix stability without encouraging excessive bone formation (13). Clearly, a complete understanding of miRNA function in different zones of the growth plate could provide additional miRNAs to target for therapeutic intervention.

Recent reviews have nicely summarized the various chemical modifications made to synthetic miRNA mimics and inhibitors that increase their stability, decrease immunogenicity, and improve cellular uptake. These reviews also discuss miRNA-based therapeutics currently in clinical trials (128, 129). Here, we limit our discussion to strategies most relevant to cartilage delivery.

The avascularity and dense extracellular matrix of cartilage pose significant barriers to drug delivery. Recent advances in nanoparticle and exosome-based delivery systems offer potential solutions, as nanoparticles and exosomes provide a vehicle for encapsulating miRNAs, enhancing their stability, and enabling their delivery to the dense cartilage matrix (130). These approaches are being optimized primarily in small animal models of osteoarthritis, where exosomes are administered via direct injection or via scaffold encapsulation for sustained release, which could improve targeting while minimizing off-target diffusion. For example, exosomes engineered with type II collagen-binding cartilage affinity peptides (CAPs) (e.g. DWRVIIPPRPSA) show enhanced retention in the joint, directing delivery of therapeutic miR-140 mimic to damaged cartilage, achieving better outcomes in preclinical osteoarthritis models (131). Additionally, novel cartilage-penetrating nanoparticles, such as CAP-PVAm-PLGA nanoparticles, were shown to penetrate ex vivo human cartilage to depths over 1000 µm, and could facilitate sustained release of therapeutic miRNAs such as miR-140 (132). Enhanced localization within the cartilage could address significant delivery challenges, potentially enabling precise treatment of growth plate injuries. Moreover, composite biomaterials incorporating nanoparticles that carry miRNA therapeutics in matrices tuned for specific mechanical properties and degradation rates may facilitate cartilage repair after bony bridge excision.

Applying miRNA-based therapies in pediatric populations presents challenges due to the sensitivity of growth plates to hormonal and genetic signals. miRNA-based interventions could inadvertently suppress normal growth processes or have off-target effects, impacting other developmental pathways. Long-term effects of altering miRNA levels in children remain uncertain, posing potential risks to immune function, systemic development, or premature growth plate closure. Therefore, precision in miRNA dosage and delivery is crucial.

Indeed, although there are miRNA-based therapeutics in clinical trials, systemic delivery is problematic due to the exposure of non-target tissues to the therapeutic. Moreover, nucleic acids can activate toll-like receptors (TLRs) and trigger an interferon response, causing side effects. Titration of an appropriate miRNA dose is also critical because exogenous miRNAs can cause unexpected responses due to their competition with endogenous miRNAs for miRNA processing machinery and RISC components. High levels of exogenous miRNAs can also engage lower affinity targets, causing additional unanticipated effects (133). Pediatric applications necessitate further research to ensure safe, effective delivery that aligns with the unique physiological demands of the growing skeleton.

While miRNA-based therapeutics, especially those targeting miR-140, hold substantial potential for cartilage repair, significant challenges remain in adapting these treatments for clinical use in growth plate disorders. Advancements in delivery platforms such as exosome engineering and nanoparticle design, combined with a thorough understanding of the molecular mechanisms by which miRNAs regulate growth plate function will lay the groundwork for innovative treatments for injury to the growth plate and other cartilage compartments (86).