MicroRNAs in osteoblast differentiation and fracture healing: From pathogenesis to therapeutic implication.

The term "fracture" pertains to the occurrence of bones being either fully or partially disrupted as a result of external forces. Prolonged fracture healing can present a notable danger to the patient's general health and overall quality of life. The significance of osteoblasts in the process of new bone formation is widely recognized, and optimizing their function could be a desirable strategy. Therefore, the mending of bone fractures is intricately linked to the processes of osteogenic differentiation and mineralization. MicroRNAs (miRNAs) are RNA molecules that do not encode for proteins, but rather modulate the functioning of physiological processes by directly targeting proteins. The participation of microRNAs (miRNAs) in experimental investigations has been extensive, and their control functions have earned them the recognition as primary regulators of the human genome. Earlier studies have shown that modulating the expression of miRNAs, either by increasing or decreasing their levels, can initiate the differentiation of osteoblasts. This implies that miRNAs play a pivotal function in promoting osteogenesis, facilitating bone mineralization and formation, ultimately leading to an efficient healing of fractures. Hence, focusing on miRNAs can be considered a propitious therapeutic approach to accelerate the healing of fractures and forestall nonunion. In this manner, the information supplied by this investigation has the potential to aid in upcoming clinical utilization, including its possible use as biomarkers or as resources for devising innovative therapeutic tactics aimed at promoting fracture healing.

Suppression of DNMT2/3 by proinflammatory cytokines inhibits CtBP1/2-dependent genes to promote the occurrence of atrophic nonunion.

Failure of bone healing after fracture often results in nonunion, but the underlying mechanism of nonunion pathogenesis is poorly understood. Herein, we provide evidence to clarify that the inflammatory microenvironment of atrophic nonunion (AN) mice suppresses the expression levels of DNA methyltransferases 2 (DNMT2) and 3A (DNMT3a), preventing the methylation of CpG islands on the promoters of C-terminal binding protein 1/2 (CtBP1/2) and resulting in their overexpression. Increased CtBP1/2 acts as transcriptional corepressors that, along with histone acetyltransferase p300 and Runt-related transcription factor 2 (Runx2), suppress the expression levels of six genes involved in bone healing: BGLAP (bone gamma-carboxyglutamate protein), ALPL (alkaline phosphatase), SPP1 (secreted phosphoprotein 1), COL1A1 (collagen 1a1), IBSP (integrin binding sialoprotein), and MMP13 (matrix metallopeptidase 13). We also observe a similar phenomenon in osteoblast cells treated with proinflammatory cytokines or treated with a DNMT inhibitor (5-azacytidine). Forced expression of DNMT2/3a or blockage of CtBP1/2 with their inhibitors can reverse the expression levels of BGLAP/ALPL/SPP1/COL1A1/IBSP/MMP13 in the presence of proinflammatory cytokines. Administration of CtBP1/2 inhibitors in fractured mice can prevent the incidence of AN. Thus, we demonstrate that the downregulation of bone healing genes dependent on proinflammatory cytokines/DNMT2/3a/CtBP1/2-p300-Runx2 axis signaling plays a critical role in the pathogenesis of AN. Disruption of this signaling may represent a new therapeutic strategy to prevent AN incidence after bone fracture.

Activin A marks a novel progenitor cell population during fracture healing and reveals a therapeutic strategy.

Insufficient bone fracture repair represents a major clinical and societal burden and novel strategies are needed to address it. Our data reveal that the transforming growth factor-ß superfamily member Activin A became very abundant during mouse and human bone fracture healing but was minimally detectable in intact bones. Single-cell RNA-sequencing revealed that the Activin A-encoding gene Inhba was highly expressed in a unique, highly proliferative progenitor cell (PPC) population with a myofibroblast character that quickly emerged after fracture and represented the center of a developmental trajectory bifurcation producing cartilage and bone cells within callus. Systemic administration of neutralizing Activin A antibody inhibited bone healing. In contrast, a single recombinant Activin A implantation at fracture site in young and aged mice boosted: PPC numbers; phosphorylated SMAD2 signaling levels; and bone repair and mechanical properties in endochondral and intramembranous healing models. Activin A directly stimulated myofibroblastic differentiation, chondrogenesis and osteogenesis in periosteal mesenchymal progenitor culture. Our data identify a distinct population of Activin A-expressing PPCs central to fracture healing and establish Activin A as a potential new therapeutic tool.

Traumatic brain injury stimulates sympathetic tone-mediated bone marrow myelopoiesis to favor fracture healing.

Traumatic brain injury (TBI) accelerates fracture healing, but the underlying mechanism remains largely unknown. Accumulating evidence indicates that the central nervous system (CNS) plays a pivotal role in regulating immune system and skeletal homeostasis. However, the impact of CNS injury on hematopoiesis commitment was overlooked. Here, we found that the dramatically elevated sympathetic tone accompanied with TBI-accelerated fracture healing; chemical sympathectomy blocks TBI-induced fracture healing. TBI-induced hypersensitivity of adrenergic signaling promotes the proliferation of bone marrow hematopoietic stem cells (HSCs) and swiftly skews HSCs toward anti-inflammation myeloid cells within 14 days, which favor fracture healing. Knockout of ß3- or ß2-adrenergic receptor (AR) eliminate TBI-mediated anti-inflammation macrophage expansion and TBI-accelerated fracture healing. RNA sequencing of bone marrow cells revealed that Adrb2 and Adrb3 maintain proliferation and commitment of immune cells. Importantly, flow cytometry confirmed that deletion of ß2-AR inhibits M2 polarization of macrophages at 7th day and 14th day; and TBI-induced HSCs proliferation was impaired in ß3-AR knockout mice. Moreover, ß3- and ß2-AR agonists synergistically promote infiltration of M2 macrophages in callus and accelerate bone healing process. Thus, we conclude that TBI accelerates bone formation during early stage of fracture healing process by shaping the anti-inflammation environment in the bone marrow. These results implicate that the adrenergic signals could serve as potential targets for fracture management.

Molecular profiling of osteoprogenitor cells reveals FOS as a master regulator of bone non-union.

Despite the advances in bone fracture treatment, a significant fraction of fracture patients will develop non-union. Most non-unions are treated with surgery since identifying the molecular causes of these defects is exceptionally challenging. In this study, compared with marrow bone, we generated a transcriptional atlas of human osteoprogenitor cells derived from healing callus and non-union fractures. Detailed comparison among the three conditions revealed a substantial similarity of callus and nonunion at the gene expression level. Nevertheless, when assayed functionally, they showed different osteogenic potential. Utilizing longitudinal transcriptional profiling of the osteoprogenitor cells, we identified FOS as a putative master regulator of non-union fractures. We validated FOS activity by profiling a validation cohort of 31 tissue samples. Our work identified new molecular targets for non-union classification and treatment while providing a valuable resource to better understand human bone healing biology.

Direct Knockdown of PDZ and LIM Domain 1 Using an Adenoviral Delivery System Accelerates Osteogenesis and Fracture Healing in Mice.

Substantial advances have been made in understanding the role of partial PDZ and LIM domain family's proteins in skeletal-related diseases. Yet, little is known about the effect of PDZ and LIM Domain 1 (Pdlim1) on osteogenesis and fracture repair. This study aimed to investigate whether direct gene delivery using an adenovirus vector carrying Pdlim1 (Ad-oePdlim1) or encoding shRNA-Pdlim1 (Ad-shPdlim1) could affect the osteogenic activity of preosteoblastic MC3T3-E1 cells in vitro, and influence the fracture healing of mice in vivo. We found that Ad-shPdlim1 transfection contributed to the calcified nodule formation in MC3T3-E1 cells. Downregulation of Pdlim1 enhanced the alkaline phosphatase activity and increased the expression of osteogenic markers (Runt-related transcription factor 2 [Runx2], collagen type I alpha 1 chain [Col1A1], osteocalcin [OCN], and osteopontin [OPN]). Further analysis indicated that Pdlim1 knockdown could activate ß-catenin signaling, as evidenced by the accumulation of ß-catenin in the nucleus and the increase levels of downstream regulators such as Lef1/Tcf7, axis inhibition protein 2, cyclin D1, and SRY-box transcription factor 9. By contrast, Pdlim1 overexpression resulted in inhibition of the osteogenic activity of MC3T3-E1 cells. In vivo, at day 3 after fracture,Ad-shPdlim1 adenovirus particles were injected into the fracture site of the femur of mice, and the process of fracture healing was evaluated by X-ray, micro-computed tomography and histological examination. Local injection of Ad-shPdlim1 promoted the early cartilage callus formation, restored bone mineral density, and accelerated cartilaginous ossification, with the upregulation of osteogenic gene (Runx2, Col1A1, OCN, and OPN) expression and activation of ß-catenin signaling. Thus, we concluded that inhibition of Pdlim1 contributed to osteogenesis and fracture healing by activating the ß-catenin signaling pathway.

[Advances on pentraxin 3 in osteoporosis and fracture healing].

Pentaxin 3 (PTX3), as a multifunctional glycoprotein, plays an important role in regulating inflammatory response, promoting tissue repair, inducing ectopic calcification and maintaining bone homeostasis. The effect of PTX3 on bone mineral density (BMD) may be affected by many factors. In PTX3 knockout mice and osteoporosis (OP) patients, the deletion of PTX3 will lead to decrease of BMD. In Korean community "Dong-gu study", it was found that plasma PTX3 was negatively correlated with BMD of femoral neck in male elderly patients. In terms of bone related cells, PTX3 plays an important role in maintaining the phenotype and function of osteoblasts (OB) in OP state;for osteoclast (OC), PTX3 in inflammatory state could stimulate nuclear factor κ receptor activator of nuclear factor-κB ligand (RANKL) production and its combination with TNF-stimulated gene 6(TSG-6) could improve activity of osteoclasts and promote bone resorption;for mesenchymal stem cells (MSCs), PTX3 could promote osteogenic differentiation of MSCs through PI3K/Akt signaling pathway. In recent years, the role of PTX3 as a new bone metabolism regulator in OP and fracture healing has been gradually concerned by scholars. In OP patients, PTX3 regulates bone mass mainly by promoting bone regeneration. In the process of fracture healing, PTX3 promotes fracture healing by coordinating bone regeneration and bone resorption to maintain bone homeostasis. In view of the above biological characteristics, PTX3 is expected to become a new target for the diagnosis and treatment of OP and other age-related bone diseases and fracture healing.

Fibulin2: a negative regulator of BMSC osteogenic differentiation in infected bone fracture healing.

Bone fracture remains a common occurrence, with a population-weighted incidence of approximately 3.21 per 1000. In addition, approximately 2% to 50% of patients with skeletal fractures will develop an infection, one of the causes of disordered bone healing. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) plays a key role in disordered bone repair. However, the specific mechanisms underlying BMSC dysfunction caused by bone infection are largely unknown. In this study, we discovered that Fibulin2 expression was upregulated in infected bone tissues and that BMSCs were the source of infection-induced Fibulin2. Importantly, Fibulin2 knockout accelerated mineralized bone formation during skeletal development and inhibited inflammatory bone resorption. We demonstrated that Fibulin2 suppressed BMSC osteogenic differentiation by binding to Notch2 and inactivating the Notch2 signaling pathway. Moreover, Fibulin2 knockdown restored Notch2 pathway activation and promoted BMSC osteogenesis; these outcomes were abolished by DAPT, a Notch inhibitor. Furthermore, transplanted Fibulin2 knockdown BMSCs displayed better bone repair potential in vivo. Altogether, Fibulin2 is a negative regulator of BMSC osteogenic differentiation that inhibits osteogenesis by inactivating the Notch2 signaling pathway in infected bone.

Long Noncoding RNA NORAD Promotes Fracture Healing through Interacting with Osteoblast Differentiation via Targeting miR-26a.

The present study was designed to evaluate the dynamic expression of lncRNA NORAD in fracture healing of patients with brittle fractures and explore the function and mechanism of NORAD in regulating osteoblastic proliferation, differentiation, and apoptosis. The expression level of NORAD was detected by quantitative real-time PCR. The proliferation, differentiation, and apoptosis of osteoblasts were analyzed by MTT assay, ELISA, and flow cytometry. Luciferase report analysis was used to confirm the interaction between NORAD and its target ceRNA miR-26a. This study showed no significant differences in serum NORAD expression on the 7th day during fracture healing in patients, but increased expression of NORAD was certified on the 14, 21, and 28 days after fixation. Overexpression of NORAD promoted the proliferation and differentiation of osteoblasts and suppressed the apoptosis of osteoblasts. miR-26a proved to be the target gene of NORAD and was inhibited by overexpression of NORAD in osteoblasts. The enhanced expression of miR-26a was negatively linked to the lessened expression of NORAD. NORAD could accelerate the proliferation and differentiation of osteoblasts and inhibit apoptosis, thereby promoting fracture healing.

Prognostic and therapeutic potential of microRNAs for fracture healing processes and non-union fractures: A systematic review.

BACKGROUND: Approximately 10% of all bone fractures result in delayed fracture healing or non-union; thus, the identification of biomarkers and prognostic factors is of great clinical interest. MicroRNAs (miRNAs) are known to be involved in the regulation of the bone healing process and may serve as functional markers for fracture healing. AIMS AND METHODS: This systematic review aimed to identify common miRNAs involved in fracture healing or non-union fractures using a qualitative approach. A systematic literature search was performed with the keywords 'miRNA and fracture healing' and 'miRNA and non-union fracture'. Any original article investigating miRNAs in fracture healing or non-union fractures was screened. Eventually, 82 studies were included in the qualitative analysis for 'miRNA and fracture healing', while 19 were selected for the 'miRNA and fracture non-union' category. RESULTS AND CONCLUSIONS: Out of 151 miRNAs, miR-21, miR-140 and miR-214 were the most investigated miRNAs in fracture healing in general. miR-31-5p, miR-221 and miR-451-5p were identified to be regulated specifically in non-union fractures. Large heterogeneity was detected between studies investigating the role of miRNAs in fracture healing or non-union in terms of patient population, sample types and models used. Nonetheless, our approach identified some miRNAs with the potential to serve as biomarkers for non-union fractures, including miR-31-5p, miR-221 and miR-451-5p. We provide a discussion of involved pathways and suggest on alignment of future research in the field.

Unveiling the Time Course Mechanism of Bone Fracture Healing by Transcriptional Profiles.

BACKGROUND: Bone fracture healing is a time-consuming and high-priority orthopedic problem worldwide. OBJECTIVE: Discovering the potential mechanism of bone healing at a time course and transcriptional level may better help manage bone fracture. METHODS: In this study, we analyze a time-course bone fracture healing transcriptional dataset in a rat model (GSE592, GSE594, and GSE1371) of Gene Expression Omnibus (GEO). RNA was obtained from female Sprague-Dawley rats with a femoral fracture at the initial time (day 3) as well as early (week 1), middle (week 2), and late (week 4) time periods, with nonfracture rats used as control. Gene Ontology (GO) functional analysis and pathway examinations were performed for further measurements of GSEA and hub genes. RESULTS: Results indicated that the four stages of bone fracture healing at the initial, early, middle, and late time periods represent the phases of hematoma formation, callus formation, callus molding, and mature lamellar bone formation, respectively. Extracellular organization was positively employed throughout the four stages. At the hematoma formation phase, the muscle contraction process was downregulated. Antibacterial peptide pathway was downregulated at all phases. The upregulation of Fn1 (initial, early, middle, and late time periods), Col3a1 (initial, early, and middle time periods), Col11a1 (initial and early time periods), Mmp9 (middle and late time periods), Mmp13 (early, middle, and late time periods) and the downregulation of RatNP-3b (initial, early, middle, and late time periods) were possible symbols for bone fracture healing and may be used as therapeutic targets. CONCLUSION: These findings suggest some new potential pathways and genes in the process of bone fracture healing and further provide insights that can be used in targeted molecular therapy for bone fracture healing.

Advances on pentraxin 3 in osteoporosis and fracture healing / 中国骨伤

Pentaxin 3 (PTX3), as a multifunctional glycoprotein, plays an important role in regulating inflammatory response, promoting tissue repair, inducing ectopic calcification and maintaining bone homeostasis. The effect of PTX3 on bone mineral density (BMD) may be affected by many factors. In PTX3 knockout mice and osteoporosis (OP) patients, the deletion of PTX3 will lead to decrease of BMD. In Korean community "Dong-gu study", it was found that plasma PTX3 was negatively correlated with BMD of femoral neck in male elderly patients. In terms of bone related cells, PTX3 plays an important role in maintaining the phenotype and function of osteoblasts (OB) in OP state;for osteoclast (OC), PTX3 in inflammatory state could stimulate nuclear factor κ receptor activator of nuclear factor-κB ligand (RANKL) production and its combination with TNF-stimulated gene 6(TSG-6) could improve activity of osteoclasts and promote bone resorption;for mesenchymal stem cells (MSCs), PTX3 could promote osteogenic differentiation of MSCs through PI3K/Akt signaling pathway. In recent years, the role of PTX3 as a new bone metabolism regulator in OP and fracture healing has been gradually concerned by scholars. In OP patients, PTX3 regulates bone mass mainly by promoting bone regeneration. In the process of fracture healing, PTX3 promotes fracture healing by coordinating bone regeneration and bone resorption to maintain bone homeostasis. In view of the above biological characteristics, PTX3 is expected to become a new target for the diagnosis and treatment of OP and other age-related bone diseases and fracture healing.

Mechanosensitive Piezo1 is crucial for periosteal stem cell-mediated fracture healing.

The biomechanical environment plays a dominant role in fracture healing, and Piezo1 is regarded as a major mechanosensor in bone homeostasis. However, the role of Piezo1 in fracture healing is not yet well characterized. In this study, we first delineated that Piezo1 is highly expressed in periosteal stem cells (PSCs) and their derived osteoblastic lineage cells and chondrocytes. Furthermore, downregulation of Piezo1 in callus leads to impaired fracture healing, while activation by its specific agonist promotes fracture healing through stimulation of PSC-modulated chondrogenesis and osteogenesis, along with accelerated cartilage-to-bone transformation. Interestingly, vascular endothelial growth factor A is upregulated after Yoda1 treatment of PSCs, indicating an indirect role of Piezo1 in angiogenesis. Mechanistically, activation of Piezo1 promotes expression of Yes-associated protein (YAP) and its nuclear localization in PSCs, which in turn increases the expression and nuclear localization of ß-catenin. In detail, YAP directly interacts with ß-catenin in the nucleus and forms a transcriptional YAP/ß-catenin complex, which upregulates osteogenic, chondrogenic and angiogenic factors. Lastly, Yoda1 treatment significantly improves fracture healing in a delayed union mouse model generated by tail suspension. These findings indicate that Piezo1 is a potential therapeutic target for fracture delayed union or nonunion.

Fracture hematoma micro-architecture influences transcriptional profile and plays a crucial role in determining bone healing outcomes.

The hematoma that forms between broken fragments of bone serves as a natural fibrin scaffold, and its removal from the defect site delays bone healing. The hypothesis of this study is that the microarchitectural and mechanical properties of the initially formed hematoma has a significant effect on the regulation of the biological process, which ultimately determines the outcome of bone healing. To mimic three healing conditions in the rat femur (normal, delayed, and non-healing bone defects), three different defect sizes of 0.5, 1.5, and 5.0 mm, are respectively used. The analysis of 3-day-old hematomas demonstrates clear differences in fibrin clot micro-architecture in terms of fiber diameter, fiber density, and porosity of the formed fibrin network, which result in different mechanical properties (stiffness) of the hematoma in each model. Those differences directly affect the biological processes involved. Specifically, RNA-sequencing reveals almost 700 differentially expressed genes between normally healing and non-healing defects, including significantly up-regulated essential osteogenic genes in normally healing defects, also differences in immune cell populations, activated osteogenic transcriptional regulators as well as potential novel marker genes. Most importantly, this study demonstrates that the healing outcome has already been determined during the hematoma phase of bone healing, three days post-surgery.

Toll-like receptor 9 (TLR9) gene deletion-mediated fracture healing in type II diabetic osteoporosis associates with inhibition of the nuclear factor-kappa B (NF-κB) signaling pathway.

Diabetes is characterized by increased fracture risk. Evidence from in vivo studies is lacking for anti-fracture strategies in diabetes. Our microarray analyses predicted association of Toll-like receptor 9 (TLR9) with both diabetes and osteoporosis, which was the focus of this work in a murine model of type II diabetic osteoporosis (T2DOP). A T2DOP model with fracture was established in TLR9 knockout (TLR9-/-) mice, which were then treated with the NF-κB signaling pathway inhibitor (PDTC) and activator (TNF-α). The obtained data suggested that TLR9 knockout augmented regeneration of bone tissues and cartilage area in the callus, and diminished fibrous tissues in T2DOP mice. Moreover, TLR9 depletion significantly affected bone mineral density (BMD), bone volume/tissue volume (BV/TV), connectivity density, trabecular number, trabecular separation and trabecular thickness, thus promoting fracture recovery. Bone morphology and structure were also improved in response to TLR9 depletion in T2DOP mice. TLR9 depletion inactivated NF-κB signaling in T2DOP mice. PDTC was found to enhance fracture healing in T2DOP mice, while TNF-α negated this effect. Collectively, these data indicate that TLR9 depletion may hold anti-fracture properties, making it a potential therapeutic target for T2DOP.Abbreviations: Diabetic osteoporosis (DOP); bone mineral density (BMD); Toll-like receptors (TLRs); type 2 diabetes (T2D); Toll-like receptor 9 (TLR9); nuclear factor-kappaB (NF-κB); streptozotocin (STZ); type 2 diabetic osteoporosis (T2DOP); Gene Expression Omnibus (GEO); Kyoto encyclopedia of genes and genomes (KEGG); pyrrolidine dithiocarbamate (PDTC); computed tomography (CT); Hematoxylin-eosin (HE); bone morphogenetic protein 7 (BMP7); analysis of variance (ANOVA).

Proteasome inhibition-enhanced fracture repair is associated with increased mesenchymal progenitor cells in mice.

The ubiquitin/proteasome system controls the stability of Runx2 and JunB, proteins essential for differentiation of mesenchymal progenitor/stem cells (MPCs) to osteoblasts. Local administration of proteasome inhibitor enhances bone fracture healing by accelerating endochondral ossification. However, if a short-term administration of proteasome inhibitor enhances fracture repair and potential mechanisms involved have yet to be exploited. We hypothesize that injury activates the ubiquitin/proteasome system in callus, leading to elevated protein ubiquitination and degradation, decreased MPCs, and impaired fracture healing, which can be prevented by a short-term of proteasome inhibition. We used a tibial fracture model in Nestin-GFP reporter mice, in which a subgroup of MPCs are labeled by Nestin-GFP, to test our hypothesis. We found increased expression of ubiquitin E3 ligases and ubiquitinated proteins in callus tissues at the early phase of fracture repair. Proteasome inhibitor Bortezomib, given soon after fracture, enhanced fracture repair, which is accompanied by increased callus Nestin-GFP+ cells and their proliferation, and the expression of osteoblast-associated genes and Runx2 and JunB proteins. Thus, early treatment of fractures with Bortezomib could enhance the fracture repair by increasing the number and proliferation of MPCs.

Macrophages and Bone Marrow-Derived Mesenchymal Stem Cells Work in Concert to Promote Fracture Healing: A Brief Review.

Bone marrow-derived mesenchymal stem cell (BMSC)-based and macrophage-based cell therapy are regarded as promising strategies to promote fracture healing because of incredible osteogenic potential of BMSCs and typical immunomodulatory function of macrophages. Apart from their respective key roles, accumulative evidence has also demonstrated the importance of cross talk between these two cell types in fracture healing process. This review takes a deep insight into the recent research progress of the synergic performance of BMSCs and macrophages by discussing not only the cells own functions but also the relevant impact factors and mechanisms (ambient microenvironment stimulus, miRNAs, etc). The aim of this review is to provide some valuable cues and technique support for the macrophage- and BMSC-related research, which will be helpful to propel BMSC/macrophage-based combined cell therapy for bone fracture treatment.

Mesenchymal stromal cell-derived septoclasts resorb cartilage during developmental ossification and fracture healing.

Developmental osteogenesis, physiological bone remodelling and fracture healing require removal of matrix and cellular debris. Osteoclasts generated by the fusion of circulating monocytes degrade bone, whereas the identity of the cells responsible for cartilage resorption is a long-standing and controversial question. Here we show that matrix degradation and chondrocyte phagocytosis are mediated by fatty acid binding protein 5-expressing cells representing septoclasts, which have a mesenchymal origin and are not derived from haematopoietic cells. The Notch ligand Delta-like 4, provided by endothelial cells, is necessary for septoclast specification and developmental bone growth. Consistent with the termination of growth, septoclasts disappear in adult and ageing bone, but re-emerge in association with growing vessels during fracture healing. We propose that cartilage degradation is mediated by rare, specialized cells distinct from osteoclasts. Our findings have implications for fracture healing, which is frequently impaired in aging humans.

Biological and molecular profile of fracture non-union tissue: A systematic review and an update on current insights.

Fracture non-union represents a common complication, seen in 5%-10% of all acute fractures. Despite the enhancement in scientific understanding and treatment methods, rates of fracture non-union remain largely unchanged over the years. This systematic review investigates the biological, molecular and genetic profiles of both (i) non-union tissue and (ii) non-union-related tissues, and the genetic predisposition to fracture non-union. This is crucially important as it could facilitate earlier identification and targeted treatment of high-risk patients, along with improving our understanding on pathophysiology of fracture non-union. Since this is an update on our previous systematic review, we searched the literature indexed in PubMed Medline; Ovid Medline; Embase; Scopus; Google Scholar; and the Cochrane Library using Medical Subject Heading (MeSH) or Title/Abstract words (non-union(s), non-union(s), human, tissue, bone morphogenic protein(s) (BMPs) and MSCs) from August 2014 (date of our previous publication) to 2 October 2021 for non-union tissue studies, whereas no date restrictions imposed on non-union-related tissue studies. Inclusion criteria of this systematic review are human studies investigating the characteristics and properties of non-union tissue and non-union-related tissues, available in full-text English language. Limitations of this systematic review are exclusion of animal studies, the heterogeneity in the definition of non-union and timing of tissue harvest seen in the included studies, and the search term MSC which may result in the exclusion of studies using historical terms such as 'osteoprogenitors' and 'skeletal stem cells'. A total of 24 studies (non-union tissue: n = 10; non-union-related tissues: n = 14) met the inclusion criteria. Soft tissue interposition, bony sclerosis of fracture ends and complete obliteration of medullary canal are commonest macroscopic appearances of non-unions. Non-union tissue colour and surrounding fluid are two important characteristics that could be used clinically to distinguish between septic and aseptic non-unions. Atrophic non-unions had a predominance of endochondral bone formation and lower cellular density, when compared against hypertrophic non-unions. Vascular tissues were present in both atrophic and hypertrophic non-unions, with no difference in vessel density between the two. Studies have found non-union tissue to contain biologically active MSCs with potential for osteoblastic, chondrogenic and adipogenic differentiation. Proliferative capacity of non-union tissue MSCs was comparable to that of bone marrow MSCs. Rates of cell senescence of non-union tissue remain inconclusive and require further investigation. There was a lower BMP expression in non-union site and absent in the extracellular matrix, with no difference observed between atrophic and hypertrophic non-unions. The reduced BMP-7 gene expression and elevated levels of its inhibitors (Chordin, Noggin and Gremlin) could potentially explain impaired bone healing observed in non-union MSCs. Expression of Dkk-1 in osteogenic medium was higher in non-union MSCs. Numerous genetic polymorphisms associated with fracture non-union have been identified, with some involving the BMP and MMP pathways. Further research is required on determining the sensitivity and specificity of molecular and genetic profiling of relevant tissues as a potential screening biomarker for fracture non-unions.

Long noncoding RNA XIST modulates microRNA-135/CREB1 axis to influence osteogenic differentiation of osteoblast-like cells in mice with tibial fracture healing.

Fracture healing is a complex event with the involvement of many cell systems, cytokines, as well as mRNAs. Herein, we report the interactions among long noncoding RNA X-inactive specific transcript (XIST)/microRNA-135 (miR-135)/cAMP response element-binding protein 1 (CREB1) axis during fracture healing. We observed increased expression of XIST in patients with long-term unhealed fracture by microarray analysis. Subsequently, a mouse model with tibial fracture and a cell model using osteoblast-like MC3T3-E1 cells were generated. The XIST overexpression during fracture healing decreased proliferation and differentiation of MC3T3-E1 cells, while silencing of XIST facilitated MC3T3-E1 cell growth. Furthermore, miR-135 targeted CREB1 and negatively regulated its expression. XIST acted as a sponge for miR-135, thereby upregulating CREB1 and promoting the activity of the TNF-α/RANKL pathway. Transfection of miR-135 inhibitor or CREB1 overexpression blocked the stimulating effects of XIST knockdown on MC3T3-E1 cell growth. Besides, specific inhibitors of the TNF-α/RANKL pathway reversed the repressive role of XIST in cell osteogenic differentiation. All in all, these findings suggest that XIST knockdown induces the differentiation of osteoblast-like cells via regulation of the miR-135/CREB1/TNF-α/RANKL axis. XIST, as a consequence, represents an attractive therapeutic strategy to accelerate fracture healing.