Identification of the microRNA transcriptome during the early phases of mammalian fracture repair
Graphical abstract
Introduction
Fracture repair is characterized by distinct morphological phases which include hematoma formation, inflammation, osteogenesis, chondrogenesis, intramembranous and endochondral ossification, and finally remodeling [1]. With the exception of inflammation and hematoma formation, the other events are essentially a recapitulation of the process of embryonic skeletal development and ultimately result in the formation of new bone that bridges the fracture site. All of these phases are interdependent and consist of the formation of distinct tissues (i.e., bone, cartilage) that require multiple cell types (stem cells, osteoprogenitors, osteoblasts, chondrocytes, fibroblasts, osteoclasts, etc.) and molecular processes (i.e., signaling and transcriptional activation and repression). It is these molecular processes that guide the regenerative potential of the fracture bone. For several decades researchers have tried to decipher these processes and the genes involved, with the hope of discovering a critical pathway that can be perturbed and lead to the enhancement of normal repair or successful acceleration the healing of difficult fractures (i.e., delayed and non-unions).
Currently, ~ 16 million fractures occur in the United States each year with 10% of them manifesting with difficulty in healing, either they are delayed or develop into a nonunion. Both of these conditions result in patient incapacity, lost wages, decreased productivity, and increased healthcare costs [2]. Despite various approaches to treat non-unions, including the more traditional ones such as exogenously applied low intensity pulsed ultrasound and electromagnetic fields [3] or more recent orthobiologics (i.e., stem cells, osteoinductive growth factors, osteoconductive scaffolds, and anabolic agents) [4], bone graft surgery remains the gold standard [5]. Thus, there is still a pressing need to identify a non-surgical means to successfully treat difficult fractures. To this end, our laboratory, as well as those of others, has embarked on a search for a molecular approach to this problem.
Previously we demonstrated the molecular complexity of fracture repair and reported on the transcriptional profiling of this process in which thousands of genes, along with signaling pathways, were observed to be activated within fracture calluses corresponding to the various phases of healing [6]. Some of these genes and processes were further investigated by our group and collaborators, and shown to be both temporally and spatially differentially expressed during fracture repair [7], [8], [9], [10], [11], [12], [13]. Despite this knowledge, we are still far away from developing a new treatment regimen. However, with the recent discovery of miRNAs as potent regulators of gene expression and downstream biological processes such as development and regeneration [14], [15], it may be possible to reach our goal. In fact, some have already proposed the development of microRNA-based drugs for the treatment of diseases [16].
miRNAs are small (18–25 nucleotides) non-coding regulatory RNAs that bind to the 3′UTR of target mRNAs leading to their degradation or translational repression [17]. Recent reports have demonstrated that miRNAs are involved in regulating many diverse processes such as cell differentiation and development [14], [15], [18], [19], [20], wound healing [21], [22], [23], especially during the inflammation and angiogenesis [24], tissue regeneration [25], [26], [27] as well as various disease states [28], [29], [30]. Most relevant to the work described herein, miRNAs have also been implicated as regulators of skeletogenesis [15], [31], [32], [33], [34]. More importantly, a number of studies have begun to report on the expression and function of miRNAs during fracture repair. miRNA-92a was shown to be down regulated following fractures and systemic administration of an antimir-92a in mice increased neovascularization, callus volume, and overall healing [35]. miRNA-21 overexpressing bone marrow stem cells that were injected locally into 4 day femoral fractures resulted in accelerated endochondral ossification, increased callus bone volume, and biomechanically stronger fractured femurs [36]. Another study investigated the presence of serum circulating miRNAs in patients with osteoporotic fractures and found that 6 miRNAs (miR-10a-5p, miR-10b-5p, miR-133b, miR-22-3p, miR-328-3p, and let-7g-5p) displayed differential levels of expression, with five subsequently shown to induce osteogenic differentiation of human mesenchymal stem cells in vitro [37]. Lastly, profiling of miRNA expression during the first 28 days of atrophic non-unions produced by periosteal cauterization in rats identified 9 highly expressed miRNAs. Further analysis of five of these miRNAs revealed that their expression peaked at PFD14 [38].
Given these data and considering the biological complexity of fracture repair, we hypothesized that a larger number of miRNAs would display differential expression. Thus, we performed a complete profiling of all known miRNAs during the early (14 days) phases of physiological fracture repair, as modeled in the mouse femur. We also reasoned that obtaining murine miRNA data would also enable us and others to utilize genetically modified mice specific to a particular candidate miRNA for future functional studies. Our data demonstrate that hundreds of miRNAs are differentially expressed and regulated during the fracture repair process. Bioinformatics analyses show many thousands of potential target mRNAs involving many biological processes. Taken together, these data provide the first complete analysis of miRNA expression during fracture repair and will aid in deciphering some of the molecular mechanisms responsible for the early phases of this tissue regenerative process.
Section snippets
Animal model
All methods and animal procedures were reviewed and approved by Stony Brook University's Institutional Animal Care and Use Committee and met or exceeded all federal guidelines for the humane use of animals in research. In addition, this study followed the guidelines established by National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and complied with the ARRIVE guidelines. Fractures were generated in the left femurs of 2 month
Differential miRNA gene expression during fracture repair
We utilized a microarray approach to identify all of the known differentially expressed miRNAs during the early phases of fracture repair (PFD 1–14), as these represent critical biological processes such as hematoma formation, inflammation, osteogenesis, chondrogenesis, and intramembranous and endochondral ossification (Fig. 1). Out of a total 1881 mouse miRNA genes present on the microarray, 959 (51%) genes were absent across all samples and were removed from further analyses while 922 (49%)
Discussion
Undoubtedly, as we have entered the realm of molecular medicine, we must have complete knowledge of the key molecules and signaling pathways if we are to devise new therapies for the enhancement of osteogenesis and overall skeletal regeneration for the treatment of skeletal trauma, diseases, and disorders. Although we currently have a wealth of data regarding molecular mechanisms of skeletal development and regeneration, our knowledge is by no means complete. In fact, we have only begun to
Conclusions
Undoubtedly, this study represents the initial stage of a much longer quest for increasing our knowledge and understanding of the molecular mechanisms responsible for physiological fracture repair. Given the fact that fracture repair recapitulates embryonic skeletal development, these data will extend beyond the realm of skeletal regeneration. Ultimately, our data dramatically increases our molecular knowledge and adds an additional dimension to understanding the complexity of the biology of
Authorship
MH conceived and designed the study, aided in animal surgeries, isolated RNA, conducted all qPCR experiments, analyzed data and wrote the manuscript. DEK designed the study and performed all animal surgeries, analyzed data and all statistical analyses and participated in writing of the manuscript. JZ performed all of the bioinformatics analyses and participated in writing of the manuscript. MH accepts responsibility for the integrity of the data analyses.
Acknowledgements
The authors gratefully acknowledge the assistance of John Schwedes of the Stony Brook University Genomics Facility and Minnie Ge for administrative support. Research reported in this publication was supported by an Institutional Support for Research and Creativity (ISRC) Grant from NYIT (MH).
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2022, Bone ReportsCitation Excerpt :In callus tissue, miR-451a was downregulated with a − 4.6 to −2.5 log2FC during D5-D14 among the top54 differentially expressed miRNAs of tibial closed fracture in mice (Bourgery et al., 2021). In another study with a femoral closed fracture in mice, miR-451a was also downregulated through D1-D14 by roughly about 55 % where contralateral femoral diaphysis was used as a control (Hadjiargyrou et al., 2016). Conversely, expression of miR-451a was higher on postfracture days 14 and 28 with dynamic expression patterns in a standard fracture healing callus in comparison to fibrous tissue of an unhealing fracture produced by periosteal cauterization in rat femurs (Waki et al., 2016).
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2021, BoneCitation Excerpt :However, surgery still remains the most effective solution for the repair of recalcitrant nonunions. As such there is a need to develop new therapeutic modalities for enhancing fracture healing and one promising approach is the modulation of miRNAs, especially since it was previously demonstrated that hundreds of miRNAs are differentially expressed during the early phases of the repair process [18]. miRNAs are a class of small (20 to 24 nucleotide) noncoding RNAs that bind to the 3′-untranslated region (UTR) of mRNA transcripts and regulate gene expression by blocking translation or directing the degradation of the target mRNA [19].
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2020, BoneCitation Excerpt :Recently, several studies described miRNAs as key regulators of bone related processes [10–13]. For instance, the local miRNA expression pattern is changed upon bone fractures in healthy animal models [14,15] and in human osteoporotic fractures [16–18]. Also, as previously demonstrated by us, miRNA transcriptome is systemically changed in a timely manner upon a critical fracture in the rat femur [19].
The multiple therapeutic applications of miRNAs for bone regenerative medicine
2017, Drug Discovery TodayCitation Excerpt :Lastly, using a diaphyseal fracture murine model, Hadjiargyrou et al. screened a large panel of miRNAs. They showed that miRNA expression and regulation and the bone-healing stage were strongly correlated, and published the first fracture repair-related miRNA transcriptome [39]. Few studies have investigated the roles of miRNAs in the interactions between tumor cells and bone microenvironment.