CSFDs are essential challenges to clinicians due to impairment in bone repair. There are many strategies to overcome these problems, such as applying osteogenic, osteoinductive, osteoconductive agents, and tissue repair factors[10]. Nowadays, many osteoinductive agents such as growth factors[31], PBM, and stem cell therapy are extensively applied to treat delayed or nonunion fractures. The combined effects of hDBM scaffold as an osteoconductive, hADS, and PBM as osteoinductive and osteogenic agents on MicroRNA expression and associated osteogenic target genes of tissues from a CSFD in rats were first estimated in this work. In the next step, for validation of molecular results, we evaluated the effects of those treatment modalities on biomechanical and stereological properties of CSFDs in the rat models.
Our results demonstrated that CSFDs of PBM, hADS, and hADS + PBM groups had more expression of MicroRNA-26 and related target genes than the CSFDs of the control group. These high expressed MicroRNA-26 and related osteogenic genes in CSFDs were also seen in the group of hADS + PBM compared to groups of PBM and hADS.
MicroRNAs do their regulatory functions by interaction with their target genes, and by these, they participate in many biological processes like bone remodeling, bone development, bone formation, and bone repair[6] [37, 12, 34]. For instance, it has been proved that the microRNA including miR- 26-a was more expressed in normal healing of bone fractures than in delayed or non-healing fractures, indicating that many microRNAs may have a primary role in regulating bone fracture healing [35, 6, 34]. These microRNAs exert their function positively or negatively on osteogenic or osteoclastogenic target genes in different processes of bone healing[31, 6].
MicroRNAs − 26a was currently known as the primary regulator in the commitment of stem cells and bone tissue growth[30, 34]. According to the recently published literature, it was proved that microRNAs-26a have an effective role in the metabolism of bone[30]. Similar pieces of the literature showed that MicroRNA-26a efficiently increases the differentiation potential of MSCs to osteogenic cells by affecting target genes in osteoporotic mice [30, 14] (Fig. 6).
In the current experiment, we explored that the microRNA-26 expression level was meaningfully increased after applying osteoinductive agents such as PBM, hADS, and PBMT + hADS in CSFD, indicating that MicroRNAs-26 and related target genes do participates in the process of PBM, hADS and hADS + PBM mediated CSFD healing. MicroRNA-26a interacts with Smad1 target genes to cause stem cells, such as bone marrow-derived stem cells (BMDSC), to develop into bone-forming cells [29]. Furthermore, several in vitro and in vivo research have shown that MicroRNA-26a boosts the capacity of MSCs to differentiate into osteogenic cells by targeting the BMP/SMAD molecular pathway and inhibits osteoclast differentiation and bone loss by targeting the BMP/SMAD molecular pathway. On the other hand, microRNAs-26 can meaningfully raise osteogenic genes like RUNX, BMP, and SMAD during the bone fracture healing process. In line with these findings, we also found that not only MicroRNAs-26a was more in bone tissues of treatment groups, in contrast, to control rats, but also, the expression of BMP/SMAD pathway genes, as MicroRNAs-26a target genes, was significantly more expressed in the groups of PBM, hADSs, and hADS + PBM compared to control group(Fig. 6).
TGF-B and BMP signaling pathways play important roles in the development and growth of skeletal systems throughout embryonic and postnatal periods [31]. These genes interact the molecules such as TGF-b/BMP ligand, receptors (as Smad-dependent signaling pathway), and independent signaling pathways such as P38, mitogen-activated protein kinase/p38 MAPK to control the differentiation of MSCs during bone and skeletal development, formation, and bone homeostasis [4, 28, 38]. Interaction between BMP and its receptor (RBMP) led to Smads phosphorylation. In return, phosphorylated R-Smads form a complex with Smad4. This complex enters the nucleus to control gene expression and increase osteogenesis [28, 33](See Fig. 6).
We showed that not only BMP signaling pathway was increased following PBM, hADS, and hADS + PBM treatment but also the RUNX2, as a marker for osteoblast differentiation and bone formation[20, 27], was significantly increased in CSFDs of hADS, PBM, and hADS + PBM groups.
Runx2, as an essential osteogenic target gene, is involved in various signal pathways related to the bone remodeling process. the stated mechanism is performed by controlling osteoblast cell differentiation [27, 20]. Runx-2 up-regulates the genes related to bone development like bone sialoprotein(BSP), osteopontin[20, 7, 32], and osteocalcin (OCN)[32]. Osterix, as a moderator for Runx-2 function during osteoblast differentiation, controls the function and differentiation of cells like osteoblasts and osteocytes. The cells affect the development, formation, and resorption of bones significantly [26, 20, 5]. This study indicates that PBM, hADS, and hADS + PBM treatment result in a significant increase not only in RUNT gene expression but also in SMAD expression. It was reported that Runx2 is more important in the motivation of Smads by BMPs as well [5].
BMP-2 and Runx2 pathways play a central role in controlling the fracture healing process. Many preclinical studies revealed that these signaling pathways improve the proliferation of MSCs, osteoblast precursor cells, and mouse primary osteoblasts cells and affect cell differentiation[32, 15]. The multiple regression analysis revealed a positive relationship between overexpression of osteogenic related genes like BMP/SMAD/RUNX and OSTREX, MicroRNAs-26, and improving biomechanical properties of CSFDs and osteoblast cell numbers in CSFDs. In other words, our study showed that PBM, hADS, and hADS + PBM treatments improve the biomechanical properties of CSFDs in comparison to the control group. Furthermore, we found that the number of osteoblasts was meaningfully increased in the hADS, PBM, and hADS + PBM groups than in the control group.
This study clarified, that MicroRNAs-26 regulates the proliferation of osteoblast cells and improves biomechanical properties of CSFDs, partially via the BMP/SMAD and RUNX2 pathway in the hADS + PBM group. The current study presented novel insights into the mechanisms of hADS + PBM treatment and the role of MicroRNAs-26 in the regulation of osteogenic gene expression of BMP/SMAD and RUNX2 in this process (Fig. 6).
We propose that hADS + PBM be pre-clinically tested on other larger animals and, if the positive effects of this treatment are confirmed, tests are performed in clinical settings to evaluate their effect on bone defects and fracture healing. Details of the cellular and molecular mechanisms related to hADS, PBM, and hADS + PBM-based promotion of CSFD should be elucidated by further research.