The role of epigenetics in medicine and in dentistry has grown impressively during the last decade and epigenomics is considered as one of the most important cutting-edge subjects of scientific discovery.1,2,3 The mainly studied epigenetic molecular mechanisms are miRNA production, DNA methylation and histone modification.

MicroRNAs (or miRNAs, or miR) are short sequences of non-coding RNAs (ncRNAs) composed by seventeen/twenty-five nucleotides.1 They are crucial in regulation of development, proliferation, differentiation, apoptosis and response to different extracellular signals and stress. They are thought to be related with the expression of osteogenic genes like RUNX2 and SMAD5.4 MicroRNA pathways regulate gene expression by inducing degradation and/or translational repression of target mRNAs.5 It is believed that more than 40% of all human genes are under the regulation of miRNAs.6 MessengerRNA can bind the promoter of specific microRNAs activating an auto-regulatory feedback loop; thus, when a specific mRNA is up-regulated, the related miRNA is also over-expressed.7 Each miRNA may target hundreds of mRNAs, and some targets are affected by multiple miRNAs. Probably, miRNAs are fundamental in the maintenance of pluripotency and undifferentiation of adult stem cells; indeed, several miRNAs appear to significantly modulate the differentiation of mesenchymal precursors in osteoblast cells, regulating the activity of transcription factors.8

In the field of Periodontology, the expression of miR-181b, mi-R19b, miR-30a, miR-let 7a, and miR-301a were found lower in healthy people if compared with subjects with periodontitis.9 MicroRNA-155 expression was found linked to the IL-1 signaling mechanisms and to the inflammatory response.10 In the field of Implant Dentistry, new titanium surfaces were functionalized to increase the expression levels of osteogenic genes.11 However, despite the interest on the use of biological activators to enhance osseointegration of dental implants, the available knowledge seems to be scarce.

Therefore, aim of the present review was to investigate in vitro and in vivo publications reporting biological effects of the use of microRNAs as activators of implant surfaces on bone formation and osseointegration.

This review was conducted following the Cook and Mulrow’s principles.12,13 A bibliographical electronic research was carried out on PubMed/Medline, selecting all potentially relevant articles dealing with the effects of the use of miRNAs as implant surfaces’ activators. All included articles were appraised, synthesized, interpreted and discussed.

The electronic search started with 91 articles and resulted in the inclusion of 5 articles. All included studies were in vitro articles investigating the genetic effects of surfaces functionalized with miRNAs compared with not functionalized surfaces on human or animal cells.14,15,16,17,18 One of the included studies had an in vivo component based on the unscrew torque of functionalized surfaces.18 Details of the included studies are reported in Table 1.

Regarding the surfaces challenged, Song et al.16 used titanium surfaces loaded with calcium and antimiR-138; Shao et al.17 used microarc-oxidated titanium surfaces (MAO) loaded with miR122; Liu et al.18 used titanium surfaces with miR204 inhibitor conjugated with gold nanoparticles and dispersed in the polylactic-co-glycolic acid; Wang et al.14 used MAO surfaces activated with microRNA-21; Wu et al.15 used MAO surfaces with microRNA-21 and MAO surfaces with antimicroRNA-138. As regards the population cells, three authors used rat bone marrow cells15,17,18 while two used human mesenchymal stem cells.14,16 The investigated genes and proteins were alkaline phosphatase (ALP), bone morphogenetic protein (BMP), collagen type 1α1 (COL1), collagen type 3α1 (COL3), osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor (RUNX2), extracellular signal-regulated kinases (ERK), phosphorylated ERK (p-ERK), siBCL2L2, siCKIP-1, miR-29b, miR-26a, antimiR-138.

All the challenged test surfaces did not exhibit any apparent cytotoxicity, nor affected cells’ viability if compared to control samples. All articles found that test surfaces up-regulated the expression of osteogenic genes. Shao et al.17 found that the expression of RUNX2, OSX, OCN, COL1, ALP and BMP-2 was increased around surfaces loaded with miR122. Moreover, while test surfaces showed more bone-like mineralized tissues, bone lacunae, osteocytes and new blood vessels, control ones showed fibrous repair processes. Liu et al.18 reported that test surfaces induced higher expression of BMP, OPG, ALP, RUNX2 and COL1, and that reached higher level of osseointegration evaluated with unscrew torque. Song et al.16 reported less focal adhesion kinase signaling and miR-138 expression in test surfaces. Wang et al.14 found an increased activity for COL1, COLIII, RUNX2, OPN and OCN on titanium surfaces functionalized with miRNA-21. Wu et al.15 found that the expression of BMP, OCN, OSX and RUNX2 was higher for MAO surfaces linked to antimiRNA-138; that the ALP expression was greater for MAO surfaces with miRNA-29b; and that the expression of collagen was also influenced by the passing of time with higher values at day 7 for surfaces with miRNA-29b and higher values at day 14 for surfaces with antimiRNA- 138. Moreover, the functionalized surfaces inducted a greater amount of secreted collagen and mineralized nodules.

MicroRNAs are a group of non-coding RNAs which contribute to epigenetic processes by preventing transcription of RNA from DNA or by destroying mRNAs after they are produced.1 Application of the modern knowledge in the field of epigenetics could be helpful to develop new surfaces able to reduce bone resorption, to stimulate bone deposition and to improve the osseointegration. Therefore, aim of the present study was to review all the available articles comparing the biological effects of not- functionalized implant surfaces with surfaces functionalized with miRNAs or antimiRNAs.

Interestingly, microarc oxidated surfaces were a common choice for control groups. Micro-rough and nano-rough surfaces proved to facilitate interactions with bone cells19,20 and, in particular, the microarc oxidated titanium surfaces (MAO) showed to have favourable effects on implant osseointegration through the induction of apatite deposition and promotion of osteoblast functions.21 However, improvements in implant topography alone could be not enough to stimulate the maximum osteogenic potential. An effective strategy for promoting an even better osseointegration involves the loading of advantageous biomolecules on implant surfaces to stimulate cellular differentiation, migration, gene expression and deposition of extracellular matrix.22,23,24 Even though there are available studies investigating the loading of oligonucleotides or peptides25,26 only a few studies investigated miRNAs. Articles challenging small-interfering RNAs (siRNA) observed a 30−45% reduction in cell viability27 and this evidence may support the idea that miRNAs are more biologically friendly than siRNAs. At present, the miRNAs used as activators for implant surfaces are miR122, miR-21, miR-29b, antimiR204 and antimiR138.

MicroRNA-122 is one of the main regulators of osteogenic differentiation of bone marrow mesenchymal stem cells through the alteration of the ERK signaling pathway.28 MicroRNA-21 is highly expressed during the osteogenic differentiation to repress stemness maintenance in osteoblasts.29 MicroRNA-21 exert its osteogenic function through the stabilization and accumulation of β-catenin, interfering with the PI3K-AKT-GSK3β pathway.30 Its overexpression accelerate osteogenesis in both human umbilical cord mesenchymal stem cells and human bone marrow MSCs.30 MicroRNA-29b has been demonstrated to be a phenotype regulator of osteoblasts by targeting anti-osteogenic factors such as histone deacetylase 4 and modulating bone extracellular matrix proteins.31 AntimiRNA-204 inhibit the effect of microRNA-204 as a negative regulator of RUNX2 during the osteogenic differentiation of mesenchymal stem cells.32 AntimiR-138 has been shown to enhance in vivo bone formation by inhibiting miRNA-138.33 Indeed, downregulating endogenous levels of miRNAs that play a negative role in osteogenesis has also been demonstrated to promote osteogenesis.34

Gene expression of several osteogenic genes was evaluated and found influenced by the use of miRNAs. Runt-related transcription factor 2 (RUNX2) is a transcription factor that regulates the differentiation of osteoblasts. It plays a major role in the synthesis of bone extracellular matrix proteins, including collagen type I, osteopontin, bone sialoprotein, osteocalcin, fibronectin, and other factors.35 Osteoprotegerin (OPN) is a mediator of cell-matrix and matrix-matrix cohesion during the formation, turnover and repair of normal and pathological mineralized tissues. The production of OPN is one of the earliest and latest, secretory activities of the osteoblast lineage and this activity manifests itself morphologically as a cement line or a lamina limitans at bone matrix interfaces.36 Osteocalcin (OCN) secreted by mature osteoblast is the non-collagenous bone matrix protein. The exact function of OCN still remain unclear, however, it is the determinant of bone formation and could regulate the rate and direction of mineral formation.35 Bone morphogenic proteins (BMP) promote differentiation of mesenchymal stem cells in osteoblasts and stimulate osteogenesis in mature osteoblasts.37,38 Osterix (Osx) is an osteoblast-specific transcription factor fundamental for bone formation and osteoblast differentiation. Osx, downstream of RUNX2, is specifically expressed in osteoblasts and chondrocytes.39,40,41,42 Alkaline phosphatase (ALP) is a glycoprotein that catalyzes the hydrolysis of phosphate monoesters at basic pH values. It is divided into 4 isoenzymes, the bone isoenzyme in involved in mammalian bone calcification.43 Type I collagen (COL) is the major constitutive protein of bone tissue. It has satisfactory biocompatibility and has the function to induce the bone formation.44

Because of the exiguous number of existing articles, further studies are required for a better comprehension of the effects induced by implants activated with miRNAs on osseogenetic genes. At present, only studies with an indirect evidence are available. Besides, if the increased expression of osseogenetic genes caused by miRNAs could lead to a high rate of osseointegration in human still remains undocumented. Despite the small amount of data, all the included studies agreed that implant surfaces functionalized with miRNAs or antimiRNAs stimulated a higher expression of osseogenic genes, higher differentiation and higher deposition of collagen and mineralized nodules.

The present review confirmed the effectiveness of miRNAs loaded to titanium implant surfaces in stimulating the expression of genes related to osteoblasts differentiation, osteogenesis, osseointegration and reparation of mineralized tissues. More studies are needed for a complete comprehension of the effects induced by miRNAs on osteoblast precursors. Further studies are required to define the role of time and to investigate the effects of miRNA functionalized surfaces with in vivo models. The knowledge in the field of epigenetics could be used to develop new miRNA activated surfaces, genetically and biologically friendly, able to enhance bone deposition.