Functionalizing biomaterials for osseointegration – microRNAs at the crossroads of bone formation and immune modulation

Abstract

MicroRNAs (miRNAs) play an important role in post-transcriptional gene regulation by binding to messenger RNA and thereby modulating protein synthesis and affecting cellular functions such as proliferation and apoptosis. In bone biology, miRNAs influence osseointegration, a process vital to the long-term success of orthopaedic implants. This process involves coordinated phases of inflammation, bone formation, and remodelling, which are regulated by miRNA-mediated signalling networks. miRNAs contribute to the regulation of osteoblast and osteoclast activity, as well as macrophage-mediated immune responses, thereby influencing bone regeneration and integration with implant surfaces. This review summarizes current insights into the role of miRNAs at the bone–implant interface, focusing particularly on their involvement in immune modulation and bone tissue formation. Furthermore, it explores emerging strategies for the local delivery of miRNAs to enhance implant integration, highlighting their therapeutic potential in improving orthopaedic outcomes.

Cite this article: Bone Joint Res 2026;15(7):864–879.

Article focus

• Description of the dynamic biology of the bone-implant interface and its importance for successful osseointegration.

• Summary of current knowledge on the role of microRNAs (miRNAs) in regulating osteogenesis, osteoimmunomodulation, and bone regeneration.

• Evaluation of local delivery strategies for miRNAs and their potential to enhance implant integration.

Key messages

• The bone-implant interface is heterogeneous and influenced by osteogenic, osteoclastic, and immune responses, all of which are tightly regulated by miRNAs.

• Specific miRNAs can either promote or inhibit bone formation and immune modulation, making them potential targets for regulating osseointegration.

• Localized delivery systems (e.g. nanocarriers, injectable hydrogel, and 3D scaffolds) represent promising miRNA-based platforms for improving implant integration.

Strengths and limitations

• This review brings together current insights into the role of miRNAs in bone formation and immune regulation, and connects them to innovative delivery strategies with potential for clinical translation.

• Most studies remain at the in vitro or preclinical stage, with limited evidence from in vivo or clinical settings. Variability in experimental designs, delivery approaches, and biomaterial platforms makes cross-study comparison difficult.

• More targeted, implant-oriented research is needed to determine the effectiveness of miRNA therapies in diverse bone environments, including normal and pathological conditions.

Introduction

MicroRNAs (miRNAs) are small non-coding RNA fragments, typically 18 to 22 nucleotides in length, that play a critical role in orchestrating post-transcriptional gene expression. By binding to the 3' untranslated regions (UTRs) of target messenger RNAs (mRNAs), they regulate mRNA degradation or translation, thereby modulating protein synthesis.^1,2 Studies have suggested that miRNAs traffic between different subcellular compartments to regulate translation, transcription, and post-transcriptional levels.^3,4 Post-transcriptional modulation of gene expression by miRNAs is a fundamental process in cell biology, influencing various cellular functions such as proliferation, differentiation, apoptosis, and metabolism.⁵ miRNAs also play an important role in the regulation of osseointegration.^6,7 Osseointegration, or the integration of biomedical implants with bone, is essential to the durability of orthopaedic prostheses.⁸ The global orthopaedic implant market is projected to grow at a compound annual rate of 5.7% from 2026 to 2034, reaching US$ 102.29 billion.⁹ This underscores the importance of achieving reliable osseointegration for the long-term success of implants. Aseptic loosening and infection are two major causes of implant failure.^10-12 Implant failure often necessitates a second surgery and reimplantation, which prolongs the patient’s recovery period and causes tremendous distress and inconvenience.^10,11

Osseointegration involves three key phases: the inflammatory phase, the bone formation phase, and the remodelling phase. Several crucial biological processes, such as inflammation, vasculogenesis, extracellular matrix (ECM) remodelling, and bone formation, occur in a well-orchestrated manner during the different stages of osseointegration, along with their related signalling pathways.¹³ During osteoimmunomodulation and resolution of the inflammation at the bone-implant interface, a microenvironment largely governed by macrophages, miRNAs play a key regulatory role. They modulate immune cell activity, macrophage polarization, and inflammatory cytokine expression.^14-16 Bone formation and resorption at the implant site involve a dynamic and reciprocal communication between bone and immune cells which goes beyond the classical osteoblast-osteoclast crosstalk.^17,18 Furthermore, miRNAs synchronize the expression of key transcription factors and signalling pathways that are essential for bone formation and function of osteoblasts and osteoclasts.^19,20 This review begins with a brief description of the dynamic and heterogeneous nature of the orthopaedic bone-implant interface. It emphasizes the complex biological factors that contribute to variability at implant sites and influence integration outcomes. In the second section, miRNA-mediated regulation of osteogenesis and immune signalling is highlighted, drawing primarily on in vitro studies and bone repair/regeneration models. The third section explores local application methods of miRNAs and presents targeted strategies for improving bone-implant integration.

Bone-implant interface

The bone-implant interface is a dynamic and heterogeneous environment characterized by a complex interplay of biological and material factors that influence the success of implant integration.²¹ This section focuses on the factors contributing to the heterogeneity of implant sites. Figure 1 illustrates the sequence of cellular events at the bone-implant interface.

Fig. 1.

Schematic representation of the sequence of events and subsequent cellular responses during osseointegration. The immediate biological response after implant placement is haemostasis. Within hours, a blood clot forms at the implant site, and a fibrin network develops. This network provides a temporary matrix for cell migration. Inflammatory cells are rapidly recruited to the bone-implant interface. Neutrophils are the first responders and subsequently attract monocytes, which differentiate into macrophages. Initially, macrophages exhibit a pro-inflammatory M1 phenotype, clearing debris and potential pathogens. As healing progresses, the macrophages polarize toward an anti-inflammatory M2 phenotype. This supports tissue repair, angiogenesis, and the recruitment of osteoprogenitor cells. The osteoblast precursors then differentiate into osteoblasts, which initiate the deposition of the extracellular matrix (ECM) and form new bone around the implant. Meanwhile, osteoclasts resorb necrotic or damaged bone to facilitate bone turnover and remodelling. These processes are essential for achieving stable and functional osseointegration. Created using BioRender.com.

Tissue injury is the initial result of implant placement during the surgical procedure. The first phase of healing begins with haemostasis, which involves the accumulation of coagulation factors on the surface of the implanted biomaterial. The formation of a fibrin matrix thereafter serves as a provisional scaffold for cellular migration.^13,22 Within 24 to 48 hours after implantation, neutrophils dominate the initial inflammatory response, followed by recruiting monocytes and macrophages. Their behaviour is influenced by biomaterial cues. The number of macrophages correlates with neutrophil abundance, and macrophage polarization is likewise shaped by neutrophil activity.^22,23 The macrophage population is highly heterogeneous, and according to single-cell transcriptomic studies,^24,25 the activation of macrophages extends beyond the M1 (classically activated macrophages)/M2 (alternatively activated macrophages) framework. They are not strictly binary, and can adopt mixed or transitional states depending on time and microenvironmental cues. However, for descriptive purposes, they can be broadly classified into two phenotypes: pro-inflammatory (M1) and anti-inflammatory (M2).^26-28 During the early stage of implantation, the predominant polarized macrophages are M1. In later stages, macrophages polarized to the M2 phenotype promote tissue repair. Proper osteoimmunomodulation thus orchestrates the inflammatory phase towards osseointegration. This also highlights the importance of the immunomodulatory properties of biomaterials in transitioning the response from a pro-inflammatory state to a pro-healing state.^29-32 In addition, macrophages secrete cytokines such as bone morphogenetic protein (BMP)-2, BMP-4, and transforming growth factor β-1 (TGF-β1) to support osteoblast proliferation and differentiation.³³ Although macrophages participate in multiple phases of osteoimmunomodulation and bone regeneration, their specific mechanism of action remains not fully understood, underscoring the importance of the crosstalk between the macrophages and the osteoblasts during osseointegration.^34,35

The next step is the complex process of bone formation, which is mediated by osteoblasts. This process involves multiple signalling pathways and regulatory mechanisms.^36,37 Adhesion of osteoblastic cells is the key to initiating the osseointegration cascade and regulating osteoblast-biomaterial interactions. Bone marrow-derived mesenchymal stromal cells (BMSCs) are recruited to the implant site, where they undergo osteogenic differentiation and contribute to new bone formation and implant integration.^38-41 Increased expression levels of vinculin and fibronectin, followed by collagen type I, are essential for the migration of osteoblasts and their adherence during the ECM formation. The bone ECM provides a dynamic niche for cell-to-matrix and cell-to-cell interactions.^42,43 Osteoinduction is then regulated by transcription factors, i.e. runt-related transcription factor 2 (Runx2), which is the master transcription factor of osteoblast differentiation. Runx2 is an upstream modulator of SP7 (Sp7 transcription factor), which is a key gene in bone formation. SP7, also known as osterix (OSX), plays a crucial role in osteoblast maturation.⁴⁴ Additionally, expressions of osteogenic proteins including osteopontin (OPN), osteocalcin (OCN), osteonectin (ON), and alkaline phosphatase (ALP) is important for osseointegration.^45,46 Thus, proper cell coordination is critical for successful osseointegration, ensuring the stability and functionality of biomaterials/implants in the body and resulting in long-term successful clinical outcomes.

Osteoclasts play an important role in osseointegration by promoting bone remodelling. These bone-resorbing cells remove necrotic or damaged bone from around the implant site, creating an environment conducive to new bone formation and implant stability. The differentiation and activity of osteoclasts are tightly regulated by the receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) signalling pathway, which maintains a balance between bone resorption and formation.^47,48 Figure 2 illustrates some of the coordinated differentiation pathways of osteoblasts and osteoclasts that have been studied as part of miRNA research thus far. It highlights how bone formation and resorption work together to maintain bone homeostasis.

Fig. 2.

Schematic representation of the differentiation process of osteoblasts and osteoclasts, highlighting key regulatory pathways. Bone marrow-derived mesenchymal stromal cells (BMSCs) differentiate into osteoblast precursors through the activation of essential transcription factors, including runt-related transcription factor 2 (Runx2) and osterix (OSX), driven by signalling pathways such as Wnt/β-catenin and bone morphogenetic proteins (BMPs). BMP signalling activates suppressor of mother against decapentaplegic (SMAD) 1/5/8 and SMAD4 promoting osteogenic commitment. Meanwhile, Wnt/β-catenin further supports the specification of the osteoblast lineage and suppresses the commitment of multipotential BMSCs into fibroblasts, adipocytes, or chondrocytes. SMAD7 can negatively regulate both BMP and transforming growth factor β (TGF-β) signalling, thus inhibiting osteoblast differentiation. Early osteoblasts express markers such as alkaline phosphatase (ALP) and collagen type I α 1 (Col1a1). As differentiation progresses, mature osteoblasts express osteopontin (OPN), osteocalcin (OCN), and osteonectin (ON), which contribute to production of the extracellular matrix (ECM) and mineralization. Osteoblasts can further differentiate into osteocytes that are embedded within the mineralized matrix. Osteoclasts originate from haematopoietic stem cells via the monocyte/macrophage lineage and differentiate into pre-osteoclasts. Wnt/β-catenin signalling regulates osteoclastogenesis by modulating the expression of osteoprotegerin (OPG) and receptor activator of receptor activator of nuclear factor kappa-B ligand (RANKL) expression, with OPG acting as a decoy receptor for RANKL to inhibit osteoclast differentiation. The balance between RANKL and OPG ensures coordination between bone formation and resorption, thereby maintaining bone homeostasis. The pathway selection is based on the main pathways regulated by miRNA described in the following sections. Created using BioRender.com.

The following sections will present possible miRNAs that play a pivotal role in osseointegration and osteoimmunomodulation. Very few studies have investigated the role of miRNAs in osseointegration specifically, as will be introduced in the next sections.⁴⁹ However, emerging evidence suggests that certain miRNAs can improve bone-implant integration.

miRNAs modulating macrophage

Macrophages play essential roles in immune responses and tissue remodelling during osseointegration. Single-cell and spatial transcriptomic analyses indicate that macrophage activation is highly heterogeneous. Thus, the pro-inflammatory M1 and anti-inflammatory, pro-regenerative M2 phenotype classifications should be considered simplified functional references rather than definitive representations of in vivo immune states. Timely M1-to-M2 phenotype transition is essential for effective bone healing, successful biomaterial integration with bone tissue, and promoting angiogenesis during osseointegration.^26,50,51 miRNAs act as post-transcriptional regulators, shaping the behaviour of macrophages by regulating the expression of inflammatory cytokines and targeting inflammatory signalling pathways.^35,52 This section highlights the involvement of specific miRNAs in controlling macrophage activity. Several miRNAs such as miR-21, miR-155, and miR-451, are introduced, which regulate both macrophage polarization and influence bone-forming cells.

miR-21

miR-21 is one of the earliest discovered and most extensively studied miRNAs. It is recognized for its broad regulatory roles in various tissues and processes, including bone formation, heart function, cancer development, immune responses, and wound healing.^53-55 In a study using macrophages harvested from the peritoneal cavities of mice, miR-21 deficiency inhibits the expression of M1 signature genes, and favours M2 genes.⁴⁹ The crosstalk between miR-21 and the lipid mediator prostaglandin E2 (PGE2) was found to be a critical factor in macrophage polarization. PGE2 inhibits the expression of miR-21 and strongly activates signal transducer and activator of transcription 3 (STAT3) signalling, leading to enhanced expression of M2 genes. The researchers identified STAT3 as a direct target of miR-21 in macrophages.⁴⁹

miR-155

Among miRNAs, miR-155 is one of the most studied miRNAs involved in macrophage polarization and is expressed at higher levels in M1 macrophages than in M2 macrophages.^56,57 A study by Li et al⁵⁸ investigated the encapsulation of miR-155 in nanocarriers for local bone delivery and macrophage polarization. They confirmed that miR-155 promotes the polarization of RAW264.7 murine macrophages toward the M1 phenotype and improves the secretion of M1-associated pro-inflammatory cytokines, such as tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and production of inducible nitric oxide synthase (iNOS), while having little impact on the expression of anti-inflammatory cytokines. They then applied miR-21 after three days. This subsequent delivery of miR-21 facilitated a shift toward an M2-like reparative phenotype, as evidenced by increased IL-10 expression, which supports a controlled M1-to-M2 transition.⁵⁸ It is worth mentioning that, taken together, these findings indicate that the impact of miR-21 on macrophage polarization is context-dependent, varying with the cells, stimulus and experimental model. Therefore, the role of miR-21 cannot be generalized and does not support a single, pro- or anti-M2 function.

miR-451

The expression level of 109 miRNAs during polarization of M1 and M2 macrophages was investigated by Zhang et al.⁵⁹ The results showed the upregulated expression of miR-451 and several other miRNAs in lipopolysaccharides (LPS)- and interferon (IFN)-γ-stimulated mouse bone marrow-derived macrophages (BMDM). miR-451 is one of the upregulated miRNAs in M1 macrophages and is involved in the inflammatory response.^57,60 miR-451a also successfully inhibited inflammation and promoted macrophage M1-to-M2 polarization by regulating macrophage migration inhibitory factor.⁶¹ Enriched exosomes from adipose-derived stem cells containing miR-451 promoted the polarization of M1 macrophages treated with LPS/IFN-γ to the M2 phenotype, thereby reducing inflammation and enhancing bone regeneration in an in vivo rat model with skull defects. Thus, it provides evidence for the role of miR-451 in the M1-to-M2 transition of macrophages following inflammation.

miR-181 family

The study by Zhang et al⁵⁹ profiled microRNA expression in human macrophages during the process of polarization. The expression levels of miR-181a, along with those of miR-155-5p, miR-204-5p, and miR-45, were found to be elevated more than twofold in M1 compared to M2. Next-generation sequencing data showed that miR-181a-5p was significantly amplified in M1 and M2c macrophages, whereas low levels of miR-181a-5p and miR-181b-5p were observed in M2a type.⁶² It can be concluded that miR-181 is part of the regulatory network controlling macrophage polarization. However, more studies need to be carried out in this regard.

miRNAs influencing bone formation and remodelling

miRNAs regulate osteogenesis by targeting specific genes involved in osteogenic differentiation. This is not only of fundamental importance for bone formation, but also for successful osseointegration. Due to the complexity of bone formation, the number of miRNAs that coordinate cell proliferation and differentiation, bone ECM production, and matrix mineralization is predicted to be substantial, and has not yet been fully identified.^63,64 Furthermore, miRNAs have been demonstrated to influence the differentiation and activity of osteoblasts and osteoclasts, which are essential for bone resorption and remodelling, and successful implant integration.^65,66 This section introduces miRNAs that play a defined role in bone formation and remodelling.

miR-21

In bone tissue, miR-21 is one of the prominent regulators of key signalling pathways activated during osteogenesis. Overexpression of miR-21 has been shown to increase the expression of osteogenic markers ALPL, RUNX2, secreted phosphoprotein 1 (SPP1), and SP7, thereby promoting matrix mineralization and bone formation, and potentially improving the implant integration.^67-69

miR-21 plays a dual role in both bone formation and bone resorption by coupling osteoclasts and osteoblasts.^68-70 It indirectly promotes osteoclast differentiation, by modulating the RANKL/OPG balance through upregulating RANKL secretion and suppressing OPG expression. Inhibiting miR-21 increases the expression of OPG resulting in reduced osteoclastic activity.^70-72 These findings are supported by a study showing that miR-21 is upregulated during RANKL-induced osteoclastogenesis.⁷³ Among 617 murine miRNAs, miR-21 was one of those stimulated by RANKL. It has been reported that miR-21 promotes osteoclast differentiation by downregulating programmed cell death 4 (PDCD4). Reduced PDCD4 expression removes repression of c-Fos, a key transcription factor that promotes osteoclast differentiation and function. RANKL also induces c-Fos, which increases the expression of the miR-21. This creates a positive feedback loop involving c-Fos, miR-21, and PDCD4 that regulates osteoclast development.⁷³ The miR-21 knockout mice exhibited normal skeletal development, but showed increased trabecular bone mass due to impaired osteoclast function and reduced bone resorption. Despite elevated RANKL and decreased OPG levels, miR-21 deficiency inhibited osteoclast differentiation and resorptive activity by PDCD4, supporting a key pro-osteoclastic role for miR-21 in vivo.⁷⁴

Exosomes containing miR-21a-5p, which are derived from M1 macrophages, promote the osteogenic differentiation of mouse osteoblast-like cells (MC3T3-E1) by upregulating the expression of RUNX2, bone gamma-carboxyglutamate protein (BGLAP), SPP1, and BMP2. In a rat cranial defect model, the local delivery of these exosomes significantly enhanced bone regeneration, through the direct targeting of the transcription factor GATA2, as evidenced by increased bone volume, mineral density, and new bone formation.⁷⁵

miR-29 family

miR-29b, a member of the miR-29 family, is one of the most prominently expressed miRNAs during osteoblast differentiation.⁷⁶ It contributes to osteoblastogenesis by downregulating the expression of the inhibitors of osteogenic signalling pathways including histone deacetylase 4, beta-interacting protein 1, and dual-specificity phosphatase–2 proteins, and suppresses osteoclastogenesis by targeting the master regulator of osteoclast differentiation, nuclear factor of activated T Cells 1 (NFATc-1). On the other hand, the other members of this family (miR-29a and miR-29c) increase the differentiation of osteoblasts only via the promotion of Wnt signalling pathway.^76,77 miR-29b also plays a role in ECM remodelling by regulating the expression of type I, IV, and V collagens in differentiated osteoblasts.⁷⁶SPARC codes the most abundant non-collagenous ECM protein in bone (ON), and it is another target of this miRNA. However, these targets are negatively regulated by miR-29b. In this context, it has been suggested that this miRNA may act as a negative regulator to prevent excessive accumulation of ECM components during skeletal mineralization.^78,79 The miR-29 family has been observed to be upregulated during RANKL-induced osteoclast differentiation in murine models. By targeting RNA of cytoskeletal organizing factors such as CDC42 (cell division control protein 42) or nuclear factor 1 A-type, miRNA-29 enhances osteoclastogenesis and bone resorptive capacity, thereby identifying it as a positive regulator of bone resorption.⁸⁰

miR-155

The role of miR-155 in osteoblast lineage differentiation seems to be contradictory. The study by Gu et al⁸¹ showed that miR-155 negatively targets suppressor of mother against decapentaplegic 5 (SMAD5, a mediator of BMP signalling). This results in inhibitory effects on BMP-2-induced osteoblast differentiation and downregulation of ALPL expression and ALP activity in MC3T3-E1. Furthermore, miR-155 contributes to the suppression of BMP-2-induced osteogenic differentiation, a process that is mediated by TNF-α and it is mostly done by targeting the expression of the suppressor of cytokine signalling 1 (SOCS1) expression in MC3T3-E1 cells.^82,83 In contrast, in an in vitro study by Yamamura et al,⁸⁴ a potential pro-osteogenic role for miR-155-5p in a material-dependent context was investigated. Regarding the fact the roughness of the tested materials was similar, miR-155-5p was significantly upregulated in MC3T3-E1 cells cultured on titanium compared to gold and stainless steel. These cells exhibited enhanced osteogenic differentiation upon overexpression of miR-155-5p, as evidenced by increased expression of key markers such as RUNX2 and collagen type I alpha 1 chain (COL1A1). These results imply that miR-155-5p plays a role in osteoblastic activity on titanium surfaces and could facilitate osseointegration. It should be noted that the role of miR-155 in this regard also varies based on the experimental setup, and differs under different circumstances.

Both TGF-β1/SMAD4 signalling and interferon-β were shown to have the same inhibitory effect on osteoclast differentiation by upregulating miR-155. miR-155 suppresses osteoclastogenesis by directly targeting and downregulating suppressor of SOCS1 and MITF (microphthalmia-associated transcription factor), two essential regulators associated with osteoclastogenesis. These findings suggest that bone resorption is regulated via a common miR-155-mediated pathway activated by different upstream signals.^82,85

miR-451a

miR-451a exhibits a dual role in osteogenesis, with its effects varying under different cellular conditions. Low levels of miR-451a are present during osteoblast proliferation, while higher levels were observed during osteoblast differentiation and mineralization, as well as with increased RUNX2 expression and ALP activity in vitro. Furthermore, miR-451a stimulated the expression of BMP-4, promoting osteoblast differentiation both in vitro and in vivo.⁸⁶ In contrast, Lu et al⁸⁷ reported that suppressing or knocking out miR-451a promotes osteogenic differentiation by relieving its inhibitory effect on BMP-6. BMP-6 increases bone formation by regulating SMAD1/5/8 expression, and miR-451a deficiency resulted in increased bone volume and improved bone regeneration in ovariectomized mice.

miR-1224-5p

A recent study demonstrated that miR-1224-5p plays a critical regulatory role in suppressing osteoclast differentiation and bone resorption by downregulating key osteoclast markers such as tartrate-resistant acid phosphatase (TRAP), Cathepsin K, and nuclear factor of activated T cells 1 (NFATc-1). In vivo experiments using a bone defect model further revealed that miR-1224-5p expression led to a significant reduction in osteoclast numbers, an increase in trabecular bone volume, and improvement in bone microarchitecture. The present findings suggest the potential involvement of miR-1224-5p in the process of bone regeneration.⁸⁸

SMAD7 targeting miRNAs

SMAD7 negatively regulates TGF-β and BMP signalling, resulting in the suppression of osteoblast differentiation, ALP activity, and mineralization. This can potentially impair bone formation and osseointegration.^89,90 Studies have demonstrated that miR-15b negatively regulates osteoblast proliferation while promoting their differentiation. This occurs through the indirect stabilization of Runx2 protein by protecting it from Smurf1 (SMAD-specific E3 ubiquitin ligase)-mediated degradation, and by enhancing the expression of osteogenic markers via targeting SMAD7.^91-93 miR-590-5p has also been identified as a suppressor of SMAD7 expression, and it has been shown to promote osteogenic differentiation by increasing Runx2 and collagen type I expression at a gene and protein level.^94,95 Similarly, miR-21-5p can also target SMAD7 and promote osteogenesis.⁹⁶ Exosomes rich in miR-21-5p, derived from fracture patients with traumatic brain injury, promote bone healing by enhancing the proliferation and osteogenic differentiation of BMSCs. This is evident through increased levels of collagen type I, Runx2, OCN expression, ALP activity, and mineralization. In mice, these exosomes accelerate fracture repair by increasing bone volume and density, as well as angiogenesis and cell proliferation. These findings underscore the significant therapeutic potential of miR-21-5p-enriched exosomes for bone repair.

High-mobility group AT-hook 2 targeting miRNAs

Negishi et al⁹⁷ demonstrated that high-mobility group AT-hook 2 (HMGA2) is essential for osteogenesis and craniofacial bone development. However, the exact mechanism of HMGA2 remains unknown. HMGA2 knockout models exhibited reduced expression of osteoblast-related genes, particularly SP7, indicating a promotive role for HMGA2 in bone formation during bone development. However, findings related to miR-33-5p and miR-497-5p, both targeting HMGA2, do not support the results of this study.^98,99 Wang et al⁹⁸ studied miR-33-5p, a mechanosensitive miRNA, under microgravity and fluid shear stress. Fluid shear stress increased the miR-33-5p expression and decreased the HMGA2 protein levels. Increased expression levels of RUNX2, SP7, and ALPL were reported upon overexpression of miR-33-5p in vitro. However, simulated microgravity had the opposite effect and suppressed both early and late osteogenic markers. miR-497-5p also worked in favour of osteoblast differentiation by targeting HMGA2 and inhibiting the c-Jun N-terminal kinase (JNK) signalling pathway.⁹⁹

miRNAs influenced by surface topography

Surface topography, which goes beyond chemistry, has the capacity to regulate osteogenic miRNAs.¹⁰⁰ Chakravorty et al¹⁰¹ showed that titanium surface modifications alter the miRNA expression profile of osteoprogenitor cells. Among the differentially expressed miRNAs were miR-215 and miR-125b, which target key osteogenic regulators, such as RUNX1 and COL5A1, as well as SMAD2 and SMAD4. Their findings also provide insight into how implant topography promotes osteogenic differentiation by linking surface-induced miRNA modulation to the activation of TGF-β/BMP and non-canonical Wnt/Ca²⁺ pathways. Implant surface roughness has been shown to significantly enhance the osteogenic differentiation of BMSCs by downregulating miR-181d-5p expression. miR-181d-5p acts as a negative regulator of osteogenesis by inhibiting mitogen-activated protein kinase (MAPK) signalling, a pathway essential for the expression of key osteogenic markers such as Runx2, ALP, and OSX. Furthermore, the inhibition of miR-181d-5p was shown to promote osteogenic differentiation. These results imply that the micro-topography of an implant surface can modulate the local molecular environment, favouring bone formation and improving osseointegration.¹⁰² Compared to smooth surfaces, surfaces with nanotopography influenced the differentiation of BMSCs into osteoblasts. Of the 117 miRNAs that were differentially expressed in cells cultured on smooth (0 nm) and nano glass disc surfaces (100 nm), 45 were upregulated, and 72 were downregulated. Among these, miR-135b-5p targets the osteogenic genes BGLAP, RUNX2, collagen type XV alpha 1 chain (COL15A1), and SP7; miR-122-5p and miR-148b-3p target SPP1; and miR-196a-5p and miR-26b-5p regulate BMP4 and BMP2, respectively.¹⁰³ Another study investigating the effect of nanotopography revealed that nanoscale surface features guide BMSCs toward osteogenic differentiation by modulating epigenetic regulators, thereby determining the fate of these cells. This effect is mediated by miRNAs, such as miR-4448, miR-4708, and miR-4773. These miRNAs upregulated the SMAD1, SMAD4, and BMP-2 signalling pathways.¹⁰⁴

Other miRNAs

Table I and Table II summarize additional relevant studies on miRNAs. These miRNAs either promote or inhibit osteoblast or osteoclast differentiation, depending on their target genes.

Table I.

List of additional miRNAs promoting bone formation.

miRNA	Targets and/or downstream effects	Functional role	Reference
miR-133b	GNB4 (G Protein Subunit β 4) downregulation ALP, OSX, OPN, and Runx2 upregulation	Enhanced viability and reduced apoptosis of osteoblasts Possibly promoting osteogenic differentiation	105
miR-223-5p	HDAC2 (histone deacetylase 2) downregulation	Induced osteoblast differentiation	106
miR-26a-5p (M2 macrophage-derived exosome enriched miR-26a-5p)	Upregulation of ALP, Runx2, OPN, and collagen type II	Promote osteogenic differentiation of BMSCs	107
miR-27a	SQSTM1 (known as P62) downregulation	Inhibition of osteoclast-mediated bone resorption	108
miR-100-5p	FGF21 (fibroblast growth factor 21) downregulation	Inhibition osteoclastogenesis and bone resorption	109
miR-335-5p	DKK1 (Dickkopf‐related protein 1) downregulation	Activation of the Wnt/β‐catenin signalling pathway and promoted osteogenic differentiation in vitro and in vivo	110,111

ALP, alkaline phosphatase; BMSCs, bone marrow mesenchymal stromal cells; miRNA, microRNA; OPN, osteopontin; Runx2, runt-related transcription factor 2; SQSTM1, sequestosome 1.

Table II.

List of additional miRNAs inhibiting bone formation.

miRNA	Targets and/or downstream effects	Functional role	Reference
miR-133a miR-135a	Runx2 downregulation SMAD5 downregulation	Inhibition of osteoblastic activity	112
miR-206	Connexin 43 downregulation	Inhibited osteoblast differentiation	113
miR-214-3p (osteoclast-derived miR-214-3p containing exosomes)	ATF4 downregulation	Inhibited osteoblast activity in vitro and reduced bone formation in vivo	114,115
miR-23a miR-135a miR-133a, miR-137, miR-204, miR-205, miR-217, miR-218, miR-30c, miR-34c, miR-338	N/A Runx2 and SMAD5 downregulation Runx2 downregulation	Inhibited ALP activity and expression of OC and OPN during osteogenic differentiation	116
miR-23a-5p (osteoclast-derived miR-23a-5p containing exosomes)	RUNX2 downregulation	Inhibitory effect of osteoclasts on osteoblasts differentiation, maintaining the balance of bone remodelling	117
miR-25-5p	SMAD2 suppression and ERK1/2 (extracellular signal-related kinase 1 and 2) pathway	Inhibition of osteogenic differentiation of BMSCs	118
miR-31	RhoA downregulation	Essential for osteoclast maturation and matrix resorption	119
miR-34b, miR-34c	Decrease in SATB2 accumulation	Inhibited osteoblast differentiation and proliferation in vivo by decreasing cyclin D1, CDK4, and CDK6 accumulation	120
miR-34c	LGR4 downregulation	Regulation of GSK3-β and NF-κB Promoting osteoclast differentiation in vitro	121
miR-34b miR-140	Runx2 and OSX downregulation Runx2, β catenin and collagen type 1 downregulation (dual role in BMP-2 suppression and modulate BMP-2 function)	Inhibited osteoblast differentiation and osteoblast cell proliferation	122
miR628-3p	Runx2 downregulation	Possible inhibitory effect on osteogenesis	123

ALP, alkaline phosphatase; ATF4, activating transcription factor 4; BMP, bone morphogenetic proteins ; BMSCs, bone marrow mesenchymal stromal cells; CDK, cyclin-dependent kinase; GSK3-β, glycogen synthase kinase-3 beta; LGR4, leucine rich repeat containing G protein-coupled receptor 4; NF-κB, nuclear factor-kappa B; OSX, osterix; RhoA, ras homolog family member A; Runx2, runt-related transcription factor 2; SATB2, special AT-rich sequence-binding protein 2; SMAD, suppressor of mother against decapentaplegic.

miRNA delivery strategies to promote osseointegration

The success of implant integration is determined by the interface between a biomaterial and its surrounding tissue in the body. Bone formation and angiogenesis are necessary for the long-term stability of the implant. On the other hand, undesirable outcomes include fibrous encapsulation, implant corrosion, inflammation, and infection. These outcomes can result in aseptic or septic loosening of the implant, thereby compromising clinical outcomes.^13,124

As previously described, many miRNAs have an impact on key osteogenic pathways that control OSX, Runx2, and BMP/SMAD signalling, for example. Others affect the macrophage lineage and polarization.^125,126 Systemic administration of miRNAs results in their distribution throughout the body, increasing the risk of off-target effects and unintended gene regulation in non-target tissues. For instance, the systemic delivery of miRNA-34a mimics in clinical trials resulted in severe immune-related adverse effects due to the activation of numerous immune pathways.^127,128 Another major challenge with systemic miRNA delivery is the instability of naked miRNAs in the bloodstream, where they are rapidly degraded by nucleases. Local delivery methods help bypass systemic circulation, thereby enhancing miRNA stability and therapeutic efficacy. Localizing miRNAs at the bone-implant interface can improve implant osseointegration. Developing delivery systems is critical to improving the therapeutic efficacy of miRNA-based bone tissue engineering.¹²⁹ Efficient delivery can result in controlled and sustained on-target release of miRNAs, maximizing therapeutic efficacy while minimizing off-target toxicity.^126,130 For example, 3D-printable granular hyaluronic acid-collagen hydrogels were developed to deliver chemically modified RNA in a bone regeneration study. Microgels with no cytotoxic effects increased osteogenic differentiation in vitro.¹³¹

In addition to delivering pro-osteogenic miRNAs, therapeutic strategies may also require neutralizing the negative effects of miRNAs on bone formation. This can be achieved through miRNA inhibitors (antisense oligonucleotides) or miRNA sponges, such as circular RNAs or long non-coding RNAs. They can affect specific miRNAs from regulating their target genes.^132,133 The following section describes some of the miRNAs delivery strategies for bone regeneration and osseointegration (Figure 3).

Fig. 3.

Depiction of common biomaterial platforms used for miRNA delivery. Among nanocarriers, nanoparticles and exosomes protect miRNAs from degradation and cell-penetrating peptides (CPPs) facilitate their cellular uptake. Hydrogels offer a biocompatible, injectable matrix capable of sustained release of miRNA at the target site. Collagen scaffolds and extracellular matrix (ECM)-simulating electrospun nanofibres can incorporate miRNAs to promote localized gene regulation, osteogenesis, and improved tissue integration, particularly around the implant surface. Created using BioRender.com.

Nanocarriers

Nanoparticles for targeted delivery: Nanoparticles appear to be promising vectors for miRNA delivery due to their biocompatibility and protection of miRNAs from degradation. Through surface engineering, they can be targeted to specific cells such as osteoblasts.¹³⁴ Geng et al⁶⁹ showed that acid-treated titanium implants with immobilized miR-21 nanocapsules promoted osteogenesis, angiogenesis, and osteoclastic activity in both in vitro and in vivo experiments, resulting in accelerated bone remodelling and osseointegration. The miR-21 nanocapsules coating efficiently and sustainably released miR-21, enhancing the expression of osteogenic markers, stimulating mineralization, and increasing vascularization around the implant site. The coated implants demonstrated significantly improved early bone-implant contact and bonding strength in mature rabbits with implants in the distal femur and tibia compared to the untreated controls. This indicates that surfaces functionalized with miR-21 can effectively accelerate integration of titanium implants. A strontium-substituted hydroxyapatite (SrHA) nanoparticle-based coating incorporating miR-21 was developed to enhance osseointegration of titanium implants. The coating promoted osteoblast proliferation, differentiation, and mineralization in vitro, while it improved bone-implant integration and angiogenesis in a rabbit drill hole model. The synergistic effect of SrHA and miR-21 resulted in the upregulation of osteogenic genes (ALPL, RUNX2, BGLAP, and COL1A1), of the angiogenic marker cluster of differentiation 31 (CD31), and in the suppression of osteoclast differentiation.¹³⁵ Liu et al¹³⁶ designed a polyetheretherketone (PEEK) implant surface functionalized with miR-21a-5p-loaded mesoporous bioactive glass nanoparticles embedded in a chitosan shell. This system facilitated sustained release of miRNA, which promoted macrophage M2 polarization through suppression of nuclear factor-kappa B (NF-κB) signalling, and increased osteogenic differentiation of BMSCs in vitro. Using rat air-pouch and femoral drilling models, the model ultimately improved bone regeneration and osseointegration in vivo.

A biodegradable carboxymethyl chitosan coating for titanium implants with incorporated miR-29b nanocapsules enhanced bone regeneration and osseointegration. Results showed that this miR-29b-functionalized surface significantly improved cell adhesion, proliferation, and osteogenic differentiation in vitro, with increased expression of osteogenic markers such as Runx2 and ALP. In vivo, implants coated with miR-29b nanocapsules showed greater bone-implant contact and accelerated new bone formation in the rat tibial defect model compared to uncoated controls, supporting the therapeutic potential of miR-29b delivery for improving titanium implant integration.¹³⁷

Pins coated with miR-34a loaded into hydroxyapatite/mesoporous organosilicon nanoparticles (HA/MONs@miR-34a) composite were used to enhance bone fracture healing. A positive effect on differentiation and resorption of rat osteoclasts in vitro, and improved bone formation in vivo, was observed in a rat tibia fracture model.¹³⁸ In another study, titanium implants coated with miR-335-5p/lipidoid nanoparticles were used to assess the osteogenic activity in vitro. An increase in the mRNA expression of osteogenic markers such as ALPL, COL1A1, and BGLAP was observed in mouse BMSCs.¹³⁹

Exosomes: One promising method for delivering biologically active substances from implant surfaces is the use of exosomes, which have been shown to carry functional molecules such as miRNAs, proteins, and lipids to modulate cellular responses. Recent studies have demonstrated that immobilizing exosomes on titanium implants enhances osseointegration by promoting osteogenesis, angiogenesis, and immunomodulation at the bone-implant interface in rat, mouse, and rabbit models.^140-143 Engineered exosomes overexpressing miR-181b (Exo-181b) enhanced bone regeneration both in vitro and in vivo. They resolved inflammation by suppressing protein kinase C delta (PRKCD), activating phosphorylated AKT (protein kinase B), and thereby promoting M2 macrophage polarization. Secretion of IL-6 and TNF-α was significantly reduced, while secretion of IL-10, an anti-inflammatory cytokine, was increased. In vitro results also showed that Exo-181b-treated macrophages secreted higher levels of BMP-2 and vascular endothelial growth factor (VEGF), stimulating the migration and osteogenic differentiation of MSCs, which was confirmed by in vivo experiments. In vivo, titanium implants coated with a hydrogel containing Exo-181b resulted in increased M2 macrophage infiltration and reduced inflammation. These implants significantly improved new bone formation and osseointegration in a rat bone defect model.¹⁴⁴

Cell-penetrating peptides: Cell-penetrating peptides (CPPs) are short, positively charged peptides that can efficiently traverse cellular membranes. This property makes CPPs ideal for delivering drugs and biomolecules, such as anticancer/-microbial peptides, nucleic acids, and miRNAs, into cells.^145,146 The novel cell-penetrating peptide R9-LK15 could efficiently deliver miRNA-29b into BMSCs. R9-LK15/miR-29b nanocomplex showed a transfection efficiency about ten times higher than standard methods without cytotoxicity and maintained the stability of miRNA in serum for up to 24 hours. Importantly, the nanocomplexes significantly promoted osteogenic differentiation and matrix mineralization by upregulating osteogenic markers such as ALP and downregulating histone deacetylase-4 (HDAC4).¹⁴⁷ The complex of miRNA-2861 and dimeric α-helical peptide named LK has been shown to effectively deliver the miRNA into BMSCs and promote osteogenic differentiation and mineralization. Results showed an increase in the RUNX2, BGLAP, and ALPL expression, as well as enhanced calcium deposition indicated by Alizarin red staining.¹⁴⁸

Injectable hydrogels

Hydrogels are hydrophilic polymers suitable for high localization of miRNA in tissues, helping to increase transfection efficiency and reduce potential side effects due to their good biocompatibility. Hydrogel microparticles are an attractive option compared to bulk hydrogel. The use of hydrogel microparticles is beneficial due to their minimally invasive application, which involves the use of small needles for delivery.^149,150 However, they are still considered macrogels. In contrast, microgels are colloidal networks in the nanometre range. With a higher surface-to-volume ratio than macrogels, they differ from macrogels even though they have the same internal gel structure. Microgels combined with encapsulated therapeutic factors provide injectable composites.^151,152

To stimulate bone regeneration, cholesterol-modified miR-26a was covalently patterned onto the fibres of an injectable poly (ethylene glycol) (PEG) hydrogel. In vitro, increased ALP activity and promoted calcium nodule deposition were observed. The hydrogel, produced via click chemistry, released the cholesterol-modified-miR-26a complex in a controlled manner after being injected into a rat bone defect model. This resulted in the repair of critical skull defects and increased osteogenesis.¹⁵³ A complex of miR-26a and CPP called RALA was produced, with the aim of repairing bone defects. The RALA/miR-26a nanoparticles efficiently transfect BMSCs in vitro and enhance osteoblastic protein markers, such as OPN and OCN, thereby promoting mineralization. In a rat calvarial defect model, nanoparticles delivered via an injectable, thermo-responsive chitosan hydrogel showed increased bone volume on avarage by 39.8% and 41.5% at four and eight weeks, respectively, compared to the control group. This group also exhibited improved mechanical properties compared to the untreated defect group of this in vivo model at week eight.¹⁵⁴ These studies demonstrated clear effects on bone regeneration. However, the effects on osseointegration have yet to be investigated.

3D scaffolds

Collagen-based scaffolds: Collagen-based scaffolds are commonly employed in orthopaedic tissue engineering.^155-158 Collagen is a major component of the ECM in the human body and is therefore considered the most promising biomaterial, with excellent biocompatibility and a wide range of applications in tissue engineering and regeneration. A collagen-nanohydroxyapatite scaffold was combined with a RALA nanoparticle-based miR-26a complex. In vitro, this complex efficiently transfected BMSCs, enhancing vascular endothelial growth factor (VEGF) secretion, ALP activity, and mineralization in both 2D and 3D cultures. In vivo, its implantation into rat calvarial defects resulted in the formation of highly mineralized and vascularized bone tissue, demonstrating the scaffold’s dual angiogenic and osteogenic potential for bone repair.¹⁵⁹

A multifunctional collagen scaffold with antimicrobial nanoparticles (copper-doped bioactive glass) and miR-138 inhibitor was developed by Sadowska et al¹⁶⁰ to stimulate osteogenesis. The scaffold provided a favourable osteoimmunomodulatory environment to inhibit infection and also promote osteogenesis in vitro and in vivo in a rat defect model. In addition, the angiogenic and vasculogenic potential of the scaffold was reported by using the ex ovo, shell-less chicken embryo model. It is worth mentioning that in another study with the same miR-138 inhibitor, multilayered chitosan-miRNA functionalized microporous titanium implants have been shown to enhance osteogenic activity. The application of the biofunctionalized titanium implant resulted in the promotion of osteogenic differentiation, which consequently led to enhanced ECM mineralization and improved osseointegration in vivo in a rat model.¹⁶¹ An in vitro study by Balagangadharan et al¹⁶² demonstrated that chitosan/nano-hydroxyapatite/nano-zirconium dioxide (CS/nHAp/nZrO2) scaffolds loaded with miR-590-5p significantly enhanced osteogenic differentiation in mouse MSCs.

Electrospun nanofibres: Electrospinning produces nanofibres with high surface-to-volume ratios, varying topographies, and fibre diameters. As a result, these nanofibres can be employed as a scaffold to simulate bone ECM to facilitate cellular adhesion and migration. A combination of an electrospun fibrous network with miRNA mimics activated the expression of osteogenic markers and enhanced bone formation both in vitro and in vivo.^163-165 Nanofibrous scaffolds prepared from poly(ε-caprolactone)-poly(citrate-siloxane) that were activated with osteogenic miR-5106 nanocomplexes significantly increased the osteogenic differentiation of BMSCs in vitro and the regeneration of new bone tissue in a rat cranial defect model in vivo.¹⁶⁶ Novel 3D hybrid nanofibre aerogels containing nanoparticles loaded with miR-26a enhanced VEGF secretion and inhibit glycogen synthase kinase-3β (GSK-3β) signalling, supporting both osteogenesis and angiogenesis. In vivo, the nanofibres promoted the healing of cranial bone defects in rats after four weeks in terms of significantly higher regenerated bone volume and bone formation area.¹⁶⁷

3D-printed hydrogel: 3D-printed hydrogel scaffold in combination with miRNA was the subject of a study by Pan et al.¹⁶⁸ A 3D plotting technique was used to generate a layer-by-layer hydrogel scaffold consisting of gelatin and alginate that was then soaked in miR-29b gold nanoparticles. The miRNA-activated scaffold showed good osteoinductive activity on BMSCs in vitro. In vivo, after a 14-day pre-culture period with human mesenchymal stem cells (hMSCs) and subcutaneous implantation in athymic nude mice, the miR-29b-activated scaffolds generated significantly more ectopic bone (micro-CT/histology; approximately four times higher bone volume than the controls), with stronger OCN and increased VEGF signals compared to the blank scaffolds.

Conclusions and future perspectives

miRNAs are emerging as powerful regulators of osteoblast differentiation and bone formation, exerting their effects through post-transcriptional control of key signalling pathways. Numerous miRNAs have been shown to modulate critical phases of osteogenesis, osteoclastogenesis, and macrophage polarization, thereby linking bone regeneration with immune modulation.^169-171 Despite increasing evidence from in vitro and in vivo studies on bone regeneration and osteoimmunology, there is limited direct evidence connecting specific miRNAs to improved osseointegration. Thus, while the application of miRNA-based therapies in the context of implant integration is biologically plausible, it remains largely hypothetical and requires further validation.^125,172 Osseointegration is a complex process that is regulated by a wide range of miRNAs. These miRNAs, many of which remain unidentified or incompletely characterized, control cell proliferation, osteoblast differentiation, ECM synthesis, and matrix mineralization.¹⁷¹ Several miRNAs, such as those of the miR-21, miR-155, miR-451, and miR-181 families, have been presented in this review, which both modulate osteoblast and osteoclast activity as well as immune cell behaviour, suggesting a coordinated regulatory network. However, their effects are not generally defined and depend on the specific conditions of the experiment. This alignment suggests that miRNAs act as shared molecular regulators at the bone-immune interface, thereby synchronizing inflammatory cues with osteogenic responses. These findings propose the potential of miRNA-based strategies to improve bone regeneration by influencing both immune responses and bone formation.

Future research should focus on well-controlled, implant-specific studies to evaluate the effects of candidate miRNAs on osseointegration in both healthy and compromised bone conditions. This should include developing and testing localized miRNA delivery systems and elucidation of their interactions with implant surface properties and immune responses. It is reasonable to envision advanced implant designs that enable site-specific miRNA delivery through hydrogels, nanoparticles, or multifunctional surface modifications. These designs would stabilize the miRNAs and control their release.

However, there are several challenges to clinical translation of delivery pharmacology, including maintaining the stability of miRNAs in vivo, achieving predictable local dosing, and controlling the release of miRNAs from the coating. Additionally, it is crucial to avoid carrier-related toxicity and immunogenicity. It is important to balance the intended complexity of a treatment with the unintended risks it may cause, since different transcripts are regulated by individual miRNAs.^173,174 Currently, no microRNA-based therapy has overcome the barriers of clinical translation and obtained FDA approval. However, there are several potential therapies that have advanced to Phase I and Phase II clinical trials for leukaemia, other types of cancers, type 2 diabetes, vascular disease, and cardiac fibrosis, among other diseases.¹⁷⁵

Furthermore, age-related alterations in miRNA expression can also impair osseointegration. For example, the downregulation of miR-494-3p in senescent osteocyte-derived exosomes suppresses osteogenesis via the phosphatase and tensin homolog/phosphoinositide 3-kinase/AKT pathway.¹⁷⁶ It is suggested that new studies focus on this aspect, given that elderly patients are the ones most likely to receive orthopaedic implants.^177-179 In addition, studies about the alteration of miRNA profile expression during an infection should be taken into consideration, as it is a clinical challenge that endangers the long-term success of implants. For example, patients with periprosthetic hip infection exhibited distinct miRNA expression profiles in their peripheral blood and periprosthetic soft-tissue compared with the control groups. This finding indicates compartment-specific miRNA regulation associated with implant-related infection.¹⁸⁰

In summary, while miRNAs are central to osteogenesis and immune regulation, their direct role in improving implant integration is promising, but not yet conclusively demonstrated. Filling this gap could lead to innovative strategies to enhance the success of orthopaedic and dental implants. Especially in the presence of known risk factors that are likely to hinder osseointegration, miRNA-based approaches could be particularly beneficial.

Data Availability

All data generated or analyzed during this study are included in the published article and/or in the supplementary material.