Osteoimmunology of Fracture Healing.

PURPOSE OF REVIEW: The purpose of this review is to summarize what is known in the literature about the role inflammation plays during bone fracture healing. Bone fracture healing progresses through four distinct yet overlapping phases: formation of the hematoma, development of the cartilaginous callus, development of the bony callus, and finally remodeling of the fracture callus. Throughout this process, inflammation plays a critical role in robust bone fracture healing. RECENT FINDINGS: At the onset of injury, vessel and matrix disruption lead to the generation of an inflammatory response: inflammatory cells are recruited to the injury site where they differentiate, activate, and/or polarize to secrete cytokines for the purposes of cell signaling and cell recruitment. This process is altered by age and by sex. Bone fracture healing is heavily influenced by the presence of inflammatory cells and cytokines within the healing tissue.

Chronic psychosocial stress compromises the immune response and endochondral ossification during bone fracture healing via ß-AR signaling.

Chronic psychosocial stress/trauma represents an increasing burden in our modern society and a risk factor for the development of mental disorders, including posttraumatic stress disorder (PTSD). PTSD, in turn, is highly comorbid with a plethora of inflammatory disorders and has been associated with increased bone fracture risk. Since a balanced inflammatory response after fracture is crucial for successful bone healing, we hypothesize that stress/trauma alters the inflammatory response after fracture and, consequently, compromises fracture healing. Here we show, employing the chronic subordinate colony housing (CSC) paradigm as a clinically relevant mouse model for PTSD, that mice subjected to CSC displayed increased numbers of neutrophils in the early fracture hematoma, whereas T lymphocytes and markers for cartilage-to-bone transition and angiogenesis were reduced. At late stages of fracture healing, CSC mice were characterized by decreased bending stiffness and bony bridging of the fracture callus. Strikingly, a single systemic administration of the ß-adrenoreceptor (AR) blocker propranolol before femur osteotomy prevented bone marrow mobilization of neutrophils and invasion of neutrophils into the fracture hematoma, both seen in the early phase after fracture, as well as a compromised fracture healing in CSC mice. We conclude that chronic psychosocial stress leads to an imbalanced immune response after fracture via ß-AR signaling, accompanied by disturbed fracture healing. These findings offer possibilities for clinical translation in patients suffering from PTSD and fracture.

From benchtop to clinic: a translational analysis of the immune response to submicron topography and its relevance to bone healing.

Proper regulation of the innate immune response to bone biomaterials after implantation is pivotal for successful bone healing. Pro-inflammatory M1 and anti-inflammatory M2 macrophages are known to have an important role in regulating the healing response to biomaterials. Materials with defined structural and topographical features have recently been found to favourably modulate the innate immune response, leading to improved healing outcomes. Calcium phosphate bone grafts with submicron-sized needle-shaped surface features have been shown to trigger a pro-healing response through upregulation of M2 polarised macrophages, leading to accelerated and enhanced bone regeneration. The present review describes the recent research on these and other materials, all the way from benchtop to the clinic, including in vitro and in vivo fundamental studies, evaluation in clinically relevant spinal fusion models and clinical validation in a case series of 77 patients with posterolateral and/or interbody fusion in the lumbar and cervical spine. This research demonstrates the feasibility of enhancing biomaterial-directed bone formation by modulating the innate immune response through topographic surface features.

Reduced Terminal Complement Complex Formation in Mice Manifests in Low Bone Mass and Impaired Fracture Healing.

The terminal complement complex (TCC) is formed on activation of the complement system, a crucial arm of innate immunity. TCC formation on cell membranes results in a transmembrane pore leading to cell lysis. In addition, sublytic TCC concentrations can modulate various cellular functions. TCC-induced effects may play a role in the pathomechanisms of inflammatory disorders of the bone, including rheumatoid arthritis and osteoarthritis. In this study, we investigated the effect of the TCC on bone turnover and repair. Mice deficient for complement component 6 (C6), an essential component for TCC assembly, and mice with a knockout of CD59, which is a negative regulator of TCC formation, were used in this study. The bone phenotype was analyzed in vivo, and bone cell behavior was analyzed ex vivo. In addition, the mice were subjected to a femur osteotomy. Under homeostatic conditions, C6-deficient mice displayed a reduced bone mass, mainly because of increased osteoclast activity. After femur fracture, the inflammatory response was altered and bone formation was disturbed, which negatively affected the healing outcome. By contrast, CD59-knockout mice only displayed minor skeletal alterations and uneventful bone healing, although the early inflammatory reaction to femur fracture was marginally enhanced. These results demonstrate that TCC-mediated effects regulate bone turnover and promote an adequate response to fracture, contributing to an uneventful healing outcome.

Distinct Effects of IL-6 Classic and Trans-Signaling in Bone Fracture Healing.

Bone healing is a complex process with closely linked phases of inflammation, regeneration, and remodeling. IL-6 may crucially regulate this process; however, the underlying mechanisms are unclear. IL-6 signals are transmitted via the transmembrane glycoprotein 130 by two distinct mechanisms: classic signaling using the membrane-anchored IL-6 receptor and trans-signaling using its soluble form. Herein, we investigated the hypothesis that IL-6 classic and trans-signaling have different functions during bone healing. To investigate fracture healing, 12-week-old C57BL/6J mice underwent a femur osteotomy. To study the function of IL-6 during the inflammatory phase, either an anti-IL-6 antibody, which inhibits IL-6 classic and trans-signaling, or soluble glycoprotein 130 fusion protein, which selectively blocks trans-signaling, was injected after 30 minutes and 48 hours. To analyze IL-6 effects in the repair phase, compounds were injected from day 7 onwards. Global IL-6 inhibition in the early phase after fracture reduced systemic inflammation, the recruitment of immune cells, and bone regeneration, resulting in delayed fracture healing. Global IL-6 inhibition during the repair phase disturbed bone formation and remodeling. In contrast, inhibition of IL-6 trans-signaling exerted minor effects on the immune response and did not influence bone repair, suggesting that the classic pathway accounts for most of the effects observed after global IL-6 inhibition. Our results reveal that IL-6 classic signaling, but not IL-6 trans-signaling, is essential for bone repair.

The Role of the Immune Cells in Fracture Healing.

PURPOSE OF REVIEW: Bone fracture healing is a complex physiological process relying on numerous cell types and signals. Inflammatory factors secreted by immune cells help to control recruitment, proliferation, differentiation, and activation of hematopoietic and mesenchymal cells. Within this review we will discuss the functional role of immune cells as it pertains to bone fracture healing. In doing so, we will outline the cytokines secreted and their effects within the healing fracture callus. RECENT FINDINGS: Macrophages have been found to play an important role in fracture healing. These immune cells signal to other cells of the fracture callus, modulating bone healing. Cytokines and cellular signals within fracture healing continue to be studied. The findings from this work have helped to reinforce the importance of osteoimmunity in bone fracture healing. Owing to these efforts, immunomodulation is emerging as a potential therapeutic target to improve bone fracture healing.

[Research consortium Neuroimmunology and pain in the research network musculoskeletal diseases]. / Forschungsverbund Neuroimmunologie und Schmerz (Neuroimpa) im Forschungsnetz Muskuloskelettale Erkrankungen.

BACKGROUND: The research consortium Neuroimmunology and Pain (Neuroimpa) explores the importance of the relationships between the immune system and the nervous system in musculoskeletal diseases for the generation of pain and for the course of fracture healing and arthritis. MATERIAL AND METHODS: The spectrum of methods includes analyses at the single cell level, in vivo models of arthritis and fracture healing, imaging studies on brain function in animals and humans and analysis of data from patients. RESULTS: Proinflammatory cytokines significantly contribute to the generation of joint pain through neuronal cytokine receptors. Immune cells release opioid peptides which activate opioid receptors at peripheral nociceptors and thereby evoke hypoalgesia. The formation of new bone after fractures is significantly supported by the nervous system. The sympathetic nervous system promotes the development of immune-mediated arthritis. The studies show a significant analgesic potential of the neutralization of proinflammatory cytokines and of opioids which selectively inhibit peripheral neurons. Furthermore, they show that the modulation of neuronal mechanisms can beneficially influence the course of musculoskeletal diseases. DISCUSSION: Interventions in the interactions between the immune system and the nervous system hold a great therapeutic potential for the treatment of musculoskeletal diseases and pain.

Functional block of IL-17 cytokine promotes bone healing by augmenting FOXO1 and ATF4 activity in cortical bone defect model.

We determine the effect of interleukin (IL)-17 neutralizing antibody on new bone regeneration. Anti-IL-17 antibody promoted new bone regeneration in cortical bone defect model by augmenting FOXO1 and ATF4 activity thereby decreasing oxidative stress. Our study demonstrates the bone healing and regeneration potential of neutralizing IL-17antibody in osteoporotic fractures. INTRODUCTION: The immune system plays important role in the fracture healing process. However, fracture healing is prolonged in disorders associated with systemic inflammation. Fracture healing is decelerated in osteoporosis, condition linked with systemic inflammation. Bone regeneration therapies like recombinant human BMP2 are associated with serious side effects. Studies have been carried out where agents like denosumab and infliximab enhance bone regeneration in osteoporotic conditions. Our previous studies show the osteoprotective and immunoprotective effects of neutralizing IL-17 antibody. Here, we determine the effect of IL-17 neutralizing antibody on new bone regeneration and compare its efficacy with known osteoporotic therapies. METHODS: For the study, female BALB/c mice were ovariectomized or sham operated and left for a month followed by a 0.6-mm drill-hole injury in femur mid-diaphysis. The treatment was commenced next day onwards with anti-IL-17, anti-RANKL (Receptor activator of nuclear factor kappa-B ligand), parathyroid hormone (PTH), or alendronate for a period of 3, 10, or 21 days. Animals were then autopsied, and femur bones were dissected out for micro-CT scanning, confocal microscopy, and gene and protein expression studies. RESULTS: Micro-CT analysis showed that anti-IL-17 antibody promoted bone healing at days 10 and 21, and the healing effect observed was significantly better than Ovx, anti-RANKL antibody, and ALN, and equal to PTH. Anti-IL-17 also enhanced new bone regeneration as assessed by calcein-labeling studies. Additionally, anti-IL-17 therapy enhanced expression of osteogenic markers and decreased oxidative stress at the injury site. CONCLUSION: Overall, our study demonstrates bone healing and regeneration potential of neutralizing IL-17 antibody in osteoporotic fractures.

Effects of Aging on Fracture Healing.

PURPOSE OF REVIEW: This review summarizes research on the physiological changes that occur with aging and the resulting effects on fracture healing. RECENT FINDINGS: Aging affects the inflammatory response during fracture healing through senescence of the immune response and increased systemic pro-inflammatory status. Important cells of the inflammatory response, macrophages, T cells, mesenchymal stem cells, have demonstrated intrinsic age-related changes that could impact fracture healing. Additionally, vascularization and angiogenesis are impaired in fracture healing of the elderly. Finally, osteochondral cells and their progenitors demonstrate decreased activity and quantity within the callus. Age-related changes affect many of the biologic processes involved in fracture healing. However, the contributions of such changes do not fully explain the poorer healing outcomes and increased morbidity reported in elderly patients. Future research should address this gap in understanding in order to provide improved and more directed treatment options for the elderly population.

Osteomacs and Bone Regeneration.

PURPOSE OF REVIEW: Mounting evidence supporting the critical contribution of macrophages, in particular osteal macrophages, to bone regeneration is reviewed. We specifically examine the potential role of macrophages in the basic multicellular units coordinating lifelong bone regeneration via remodelling and bone regeneration in response to injury. We review and discuss the distinctions between macrophage and osteoclast contributions to bone homeostasis, particularly the dichotomous role of the colony-stimulating factor 1-colony-stimulating factor 1 receptor axis. RECENT FINDINGS: The impact of inflammation associated with aging and other hallmarks of aging, including senescence, on macrophage function is addressed in the context of osteoporosis and delayed fracture repair. Resident macrophages versus recruited macrophage contributions to fracture healing are also discussed. We identify some of the remaining knowledge gaps that will need to be closed in order to maximise benefits from therapeutically modulating or mimicking the function of macrophages to improve bone health and regeneration over a lifetime.

Osteoimmunology in Bone Fracture Healing.

PURPOSE OF REVIEW: In the process of bone fracture healing, inflammation is thought to be an essential process that precedes bone formation and remodeling. We review recent studies on bone fracture healing from an osteoimmunological point of view. RECENT FINDINGS: Based on previous observations that many types of immune cells infiltrate into the bone injury site and release a variety of molecules, recent studies have addressed the roles of specific immune cell subsets. Macrophages and interleukin (IL)-17-producing γδ T cells enhance bone healing, whereas CD8+ T cells impair bone repair. Additionally, IL-10-producing B cells may contribute to bone healing by suppressing excessive and/or prolonged inflammation. Although the involvement of other cells and molecules has been suggested, the precise underlying mechanisms remain elusive. Accumulating evidence has begun to reveal the deeper picture of bone fracture healing. Further studies are required for the development of novel therapeutic strategies for bone fracture.

The role of complement in trauma and fracture healing.

The complement system, as part of innate immunity, is activated immediately after trauma in response to various pathogen- and danger-associated molecular patterns (PAMPs and DAMPs), and helps to eliminate microorganisms and damaged cells. However, recent data indicate an extended role of complement far beyond pure "killing", which includes regulation of the cytokine/chemokine network, influencing physiological barriers, interaction with the coagulation cascade, and even involvement with bone metabolism and repair. Complement-induced hyper-activation and dysfunction reveal the dark side of this system, leading to complications such as sepsis, multiple-organ dysfunction, delayed fracture healing, and unfavorable outcome. Thus, the present review focuses on less known regulatory roles of the complement system after trauma and during fracture healing, rather than on its bacterial and cellular "killing functions". In particular, various complement crosstalks after trauma, including the coagulation cascade and apoptosis system, appear to be crucially involved early after trauma. Long-term effects of complement on tissue regeneration after fracture and bone turnover are also considered, providing new insights into innate immunity in local and systemic complement-driven effects after trauma.

A Pronounced Inflammatory Activity Characterizes the Early Fracture Healing Phase in Immunologically Restricted Patients.

Immunologically restricted patients such as those with autoimmune diseases or malignancies often suffer from delayed or insufficient fracture healing. In human fracture hematomas and the surrounding bone marrow obtained from immunologically restricted patients, we analyzed the initial inflammatory phase on cellular and humoral level via flow cytometry and multiplex suspension array. Compared with controls, we demonstrated higher numbers of immune cells like monocytes/macrophages, natural killer T (NKT) cells, and activated T helper cells within the fracture hematomas and/or the surrounding bone marrow. Also, several pro-inflammatory cytokines such as Interleukin (IL)-6 and Tumor necrosis factor α (TNFα), chemokines (e.g., Eotaxin and RANTES), pro-angiogenic factors (e.g., IL-8 and Macrophage migration inhibitory factor: MIF), and regulatory cytokines (e.g., IL-10) were found at higher levels within the fracture hematomas and/or the surrounding bone marrow of immunologically restricted patients when compared to controls. We conclude here that the inflammatory activity on cellular and humoral levels at fracture sites of immunologically restricted patients considerably exceeds that of control patients. The initial inflammatory phase profoundly differs between these patient groups and is probably one of the reasons for prolonged or insufficient fracture healing often occurring within immunologically restricted patients.

Isolated metaphyseal injury influences unrelated bones.

Background and purpose - Fracture healing involves different inflammatory cells, some of which are not part of the traditional bone field, such as B-cells and cytotoxic T-cells. We wanted to characterize bone healing by flow cytometry using 15 different inflammatory cell markers in a mouse model of metaphyseal injury, and incidentally discovered a previously unknown general skeletal reaction to trauma. Material and methods - A bent needle was inserted and twisted to traumatize the cancellous bone in the proximal tibia of C57/Bl6 female mice. This is known to induce vivid bone formation locally in the marrow compartment. Cells were harvested from the injured region, the uninjured contralateral tibia, and the humerus. The compositions of the immune cell populations were compared to those in untraumatized control animals. Results - Tibial metaphyseal injury led to substantial changes in the cell populations over time. Unexpectedly, similar changes were also seen in the contralateral tibia and in the humerus, despite the lack of local trauma. Most leukocyte subsets were affected by this generalized reaction. Interpretation - A relatively small degree of injury to the proximal tibia led to systemic changes in the immune cell populations in the marrow of unrelated bones, and probably in the entire skeleton. The few changes that were specific for the injury site appeared to relate to modulatory functions.

Neutrophils contribute to fracture healing by synthesizing fibronectin+ extracellular matrix rapidly after injury.

The role of inflammatory cells in bone regeneration remains unclear. We hypothesize that leukocytes contribute to fracture healing by rapidly synthesizing an "emergency extracellular matrix (ECM)" before stromal cells infiltrate the fracture hematoma (FH) and synthesize the eventual collagenous bone tissue. 53 human FHs were isolated at different time points after injury, ranging from day 0 until day 23 after trauma and stained using (immuno)histochemistry. FHs isolated within 48 h after injury contained fibronectin(+) ECM, which increased over time. Neutrophils within the early FHs stained positive for cellular fibronectin and neutrophil derived particles were visible within the fibronectin(+) ECM. Stromal cells appeared at day 5 after injury or later and collagen type I birefringent fibrils could be identified during the second week after injury. Our study suggests that neutrophils contribute to bone regeneration by synthesizing an "emergency ECM" before stromal cells infiltrate the FH and synthesize the eventual bone tissue.

TNF-α is upregulated in T2DM patients with fracture and promotes the apoptosis of osteoblast cells in vitro in the presence of high glucose.

Fracture healing is regulated by proinflammatory mediators such as tumor necrosis factor-α (TNF-α), which poses influence on the balance between bone formation and remodeling. And the diabetes is thought to contribute to the delayed diabetic fracture healing. In the present study, we examined the promotion to proinflammatory cytokines and chemokines in type 2 diabetes mellitus (T2DM) patients with bone fractures, and then evaluated the promotion to TNF-α by the high glucose treatment in human osteoblast-like MG-63 cells and the regulatory role of the promoted TNF-α on the MG-63 cell apoptosis. It was demonstrated that there were significantly-upregulated high-sensitivity C-reactive protein (hsCRP) TNF-α, IL-1ß, IL-6, IFN-γ-inducible protein 10 (IP-10) and RANTES in T2DM patients with bone fracture. And the promotion to TNF-α and IL-1ß was confirmed in vitro in both mRNA and protein levels in high glucose-treated MG-63 cells. And either TNF-α or high glucose reduced the viability of MG-63 cells, promoted apoptosis and upregulated apoptosis-associated markers, such as released cytochrome c, cleaved caspase 3 and lyzed PARP. Moreover, there was a synergistic effect between TNF-α and high glucose. The viability reduction and the apoptosis induction of MG-63 cells were significantly higher in the group with both TNF-α and high glucose treatments, than in the group with singular TNF-α treatment. In conclusion, our study demonstrated that proinflammatory cytokines and chemokines were promoted in T2DM patients with bone fracture or in osteoblasts by the high glucose stimulation. TNF-α and high glucose synergistically reduced the viability and induced the apoptosis in the osteoblast-like MG-63 cells in vitro. It implies the significant regulatory role of TNF-α in the delayed fracture healing in T2DM.

Severe muscle trauma triggers heightened and prolonged local musculoskeletal inflammation and impairs adjacent tibia fracture healing.

OBJECTIVES: Complicated fracture healing is often associated with the severity of surrounding muscle tissue trauma. Since inflammation is a primary determinant of musculoskeletal health and regeneration, it is plausible that delayed healing and non-unions are partly caused by compounding local inflammation in response to concomitant muscle trauma. METHODS AND RESULTS: To investigate this possibility, a Lewis rat open fracture model [tibia osteotomy with adjacent tibialis anterior (TA) muscle volumetric muscle loss (VML) injury] was interrogated. We observed that VML injury impaired tibia healing, as indicated by diminished mechanical strength and decreased mineralized bone within the fracture callus, as well as continued presence of cartilage instead of woven bone 28 days post-injury. The VML injured muscle presented innate and adaptive immune responses that were atypical of canonical muscle injury healing. Additionally, the VML injury resulted in a perturbation of the inflammatory phase of fracture healing, as indicated by elevations of CD3(+) lymphocytes and CD68+ macrophages in the fracture callus at 3 and 14d post-injury, respectively. CONCLUSIONS: These data indicate that heightened and sustained innate and adaptive immune responses to traumatized muscle are associated with impaired fracture healing and may be targeted for the prevention of delayed and non-union following musculoskeletal trauma.

Impaired healing of fragility fractures in type 2 diabetes: clinical and radiographic assessments and serum cytokine levels.

BACKGROUND: Diabetes induces bone alterations accompanied by altered cytokine expression patterns. These alterations lead to modified fracture healing, contributing to musculoskeletal fragility in the elderly. AIMS: We evaluated the inflammatory immune response in diabetic patients during fracture healing relative to clinical and radiographic assessments. METHODS: Fifty patients of both sexes with fragility fractures were studied: 30 diabetics (group A, mean age 73.4 ± 11.2 years) and 20 normoglycemic controls (group B, mean age 75.1 ± 16.9 years). Two subgroups comprised those with hip or wrist fragility fractures (25 and 16 patients, respectively). We evaluated serum concentrations of tumor necrosis factor α, interleukins 4 and 8, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor, and epidermal growth factor (EGF) before and at 4 and 8 weeks after surgery. We also determined the Radiographic Union Score for Hips and the Radius Union Scoring System score and applied the Physical Activity Scale for the Elderly test at the same time points. Each patient underwent bone densitometry. RESULTS: MCP-1 and EGF levels were higher in group A than in group B at 4 weeks after surgery (p > 0.05). Radiographic evaluation showed lower scores in group A (p < 0.05). The main difference between the groups was evident 4 weeks after surgery. Changes in the serum concentrations of chemotactic and angiogenic factors could explain the radiographically proved impaired fracture healing in diabetic patients. CONCLUSIONS: Fragility fracture healing is impaired in diabetic patients. Radiographic and molecular patterns confirmed that the most compromised fracture-healing phase is at 4 weeks after surgery, during callus mineralization.

Genetic and cellular evidence of decreased inflammation associated with reduced incidence of posttraumatic arthritis in MRL/MpJ mice.

OBJECTIVE: To examine the relationship between inflammation and posttraumatic arthritis (PTA) in a murine intraarticular fracture model. METHODS: Male C57BL/6 and MRL/MpJ "superhealer" mice received tibial plateau fractures using a previously established method. Mice were killed on day 0 (within 4 hours of fracture) and days 1, 3, 5, 7, 28, and 56 after fracture. Synovial tissue samples, obtained prior to fracture and on days 0, 1, 3, 5, and 7 after fracture, were examined by reverse transcription-polymerase chain reaction for gene expression of proinflammatory cytokines and chemokines. Synovial fluid and serum samples were collected to measure cytokine concentrations, using enzyme-linked immunosorbent assay. Whole joints were examined histologically for the extent of synovitis and cartilage degradation, and joint tissue samples from all time points were analyzed immunohistochemically to evaluate the distribution of interleukin-1 (IL-1). RESULTS: Compared to C57BL/6 mice, MRL/MpJ mice had less severe intraarticular and systemic inflammation following joint injury, as evidenced by lower gene expression of tumor necrosis factor α and IL-1ß in the synovial tissue and lower protein levels of IL-1α and IL-1ß in the synovial fluid, serum, and joint tissues. Furthermore, after joint injury, MRL/MpJ mice had lower gene expression of macrophage inflammatory proteins and macrophage-derived chemokine (CCL22) in the synovial tissue, and also had reduced acute and late-stage infiltration of synovial macrophages. CONCLUSION: C57BL/6 mice exhibited higher levels of inflammation than MRL/MpJ mice, indicating that MRL/MpJ mice are protected from PTA in this model. These data thus suggest an association between joint tissue inflammation and the development and progression of PTA in mice.

A distinct "repair" role of regulatory T cells in fracture healing.

Regulatory T cells (Tregs) suppress immune responses and inflammation. Here, we described the distinct nonimmunological role of Tregs in fracture healing. The recruitment from the circulation pool, peripheral induction, and local expansion rapidly enriched Tregs in the injured bone. The Tregs in the injured bone displayed superiority in direct osteogenesis over Tregs from lymphoid organs. Punctual depletion of Tregs compromised the fracture healing process, which leads to increased bone nonunion. In addition, bone callus Tregs showed unique T-cell receptor repertoires. Amphiregulin was the most overexpressed protein in bone callus Tregs, and it can directly facilitate the proliferation and differentiation of osteogenic precursor cells by activation of phosphatidylinositol 3-kinase/protein kinase B signaling pathways. The results of loss- and gain-function studies further evidenced that amphiregulin can reverse the compromised healing caused by Treg dysfunction. Tregs also enriched in patient bone callus and amphiregulin can promote the osteogenesis of human pre-osteoblastic cells. Our findings indicate the distinct and nonredundant role of Tregs in fracture healing, which will provide a new therapeutic target and strategy in the clinical treatment of fractures.