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.

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.

Time Course of Immune Response and Immunomodulation During Normal and Delayed Healing of Musculoskeletal Wounds.

Single trauma injuries or isolated fractures are often manageable and generally heal without complications. In contrast, high-energy trauma results in multi/poly-trauma injury patterns presenting imbalanced pro- and anti- inflammatory responses often leading to immune dysfunction. These injuries often exhibit delayed healing, leading to fibrosis of injury sites and delayed healing of fractures depending on the intensity of the compounding traumas. Immune dysfunction is accompanied by a temporal shift in the innate and adaptive immune cells distribution, triggered by the overwhelming release of an arsenal of inflammatory mediators such as complements, cytokines and damage associated molecular patterns (DAMPs) from necrotic cells. Recent studies have implicated this dysregulated inflammation in the poor prognosis of polytraumatic injuries, however, interventions focusing on immunomodulating inflammatory cellular composition and activation, if administered incorrectly, can result in immune suppression and unintended outcomes. Immunomodulation therapy is promising but should be conducted with consideration for the spatial and temporal distribution of the immune cells during impaired healing. This review describes the current state of knowledge in the spatiotemporal distribution patterns of immune cells at various stages during musculoskeletal wound healing, with a focus on recent advances in the field of Osteoimmunology, a study of the interface between the immune and skeletal systems, in long bone fractures. The goals of this review are to (1) discuss wound and fracture healing processes of normal and delayed healing in skeletal muscles and long bones; (2) provide a balanced perspective on temporal distributions of immune cells and skeletal cells during healing; and (3) highlight recent therapeutic interventions used to improve fracture healing. This review is intended to promote an understanding of the importance of inflammation during normal and delayed wound and fracture healing. Knowledge gained will be instrumental in developing novel immunomodulatory approaches for impaired healing.

Age-related changes to macrophages are detrimental to fracture healing in mice.

The elderly population suffers from higher rates of complications during fracture healing that result in increased morbidity and mortality. Inflammatory dysregulation is associated with increased age and is a contributing factor to the myriad of age-related diseases. Therefore, we investigated age-related changes to an important cellular regulator of inflammation, the macrophage, and the impact on fracture healing outcomes. We demonstrated that old mice (24 months) have delayed fracture healing with significantly less bone and more cartilage compared to young mice (3 months). The quantity of infiltrating macrophages into the fracture callus was similar in old and young mice. However, RNA-seq analysis demonstrated distinct differences in the transcriptomes of macrophages derived from the fracture callus of old and young mice, with an up-regulation of M1/pro-inflammatory genes in macrophages from old mice as well as dysregulation of other immune-related genes. Preventing infiltration of the fracture site by macrophages in old mice improved healing outcomes, with significantly more bone in the calluses of treated mice compared to age-matched controls. After preventing infiltration by macrophages, the macrophages remaining within the fracture callus were collected and examined via RNA-seq analysis, and their transcriptome resembled macrophages from young calluses. Taken together, infiltrating macrophages from old mice demonstrate detrimental age-related changes, and depleting infiltrating macrophages can improve fracture healing in old mice.

NLRP3 inflammasome inhibitor glyburide expedites diabetic-induced impaired fracture healing.

Localized inflammation is accompanied by the diabetic-induced fracture. The present study aims to investigate the therapeutic effects of glyburide, an NLRP3 inflammasome inhibitor, in a diabetic-induced fracture model. An animal model of diabetic-induced fracture was established and the mice were administrated with metformin or glyburide for 3 weeks. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to evaluate the relative expressions of IFN-γ, TNF-α, and IL-6. Micro-computed tomography (µCT) scanning was applied to evaluate bone callus formation. Histopathology examinations of fractured femur sections were performed using Tartrate-resistant acid phosphatase (TRAP) staining and Alcian blue and orange G staining. Bone strength was evaluated using Torsional testing. Our results showed that treatment of glyburide significantly decreased the expressions of IFN-γ, TNF-α, and IL-6 in the fracture calluses in diabetic-induced fracture model, while bone callus volume and bone volume fraction were increased. Additionally, our results also demonstrated that treatment of glyburide rescued the increase of osteoclasts in the bone-cartilage interface. Apart from decreasing a percentage of cartilage area and increasing the percentage of bone and fibrotic tissue area, treatment of glyburide increased the maximum torque and yield torque of fractures. These results implied that glyburide might be used as a potential drug candidate for diabetic-induced fracture.

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.

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.

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.

Impairment of early fracture healing by skeletal muscle trauma is restored by FK506.

BACKGROUND: Heightened local inflammation due to muscle trauma or disease is associated with impaired bone regeneration. METHODS: We hypothesized that FK506, an FDA approved immunomodulatory compound with neurotrophic and osteogenic effects, will rescue the early phase of fracture healing which is impaired by concomitant muscle trauma in male (~4 months old) Lewis rats. FK506 (1 mg/kg; i.p.) or saline was administered systemically for 14 days after an endogenously healing tibia osteotomy was created and fixed with an intermedullary pin, and the overlying tibialis anterior (TA) muscle was either left uninjured or incurred volumetric muscle loss injury (6 mm full thickness biopsy from middle third of the muscle). RESULTS: The salient observations of this study were that 1) concomitant TA muscle trauma impaired recovery of tibia mechanical properties 28 days post-injury, 2) FK506 administration rescued the recovery of tibia mechanical properties in the presence of concomitant TA muscle trauma but did not augment mechanical recovery of an isolated osteotomy (no muscle trauma), 3) T lymphocytes and macrophage presence within the traumatized musculature were heightened by trauma and attenuated by FK506 3 days post-injury, and 4) T lymphocyte but not macrophage presence within the fracture callus were attenuated by FK506 at 14 days post-injury. FK506 did not improve TA muscle isometric torque production CONCLUSION: Collectively, these findings support the administration of FK506 to ameliorate healing of fractures with severe muscle trauma comorbidity. The results suggest one potential mechanism of action is a reduction in local T lymphocytes within the injured musculoskeletal tissue, though other mechanisms to include direct osteogenic effects of FK506 require further investigation.

T and B cells participate in bone repair by infiltrating the fracture callus in a two-wave fashion.

Fracture healing is a regenerative process in which bone is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process by recruiting accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. In order to answer this question, we analyzed femoral fracture healing in mice by confocal microscopy. Surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site and were detectable predominantly at the femoral neck and knee. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralization. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby the newly forming bone. During healing B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation. Both, osteoblasts and osteoclasts were found to have direct cell-cell contact with lymphocytes, strongly suggesting a regulatory role of the immune cells specifically in the later stages of fracture healing.

Complement C3 and C5 deficiency affects fracture healing.

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3(-/-) and C5(-/-) as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3(-/-): -25%, p=0.02; C5(-/-): -20% p=0.052) and newly formed bone (C3(-/-): -38%, p=0.01; C5(-/-): -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3(-/-) mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.

Fracture healing is accelerated in the absence of the adaptive immune system.

Fracture healing is a unique biologic process starting with an initial inflammatory response. As in other regenerative processes, bone and the immune system interact closely during fracture healing. This project was aimed at further elucidating how the host immune system participates in fracture healing. A standard closed femoral fracture was created in wild-type (WT) and recombination activating gene 1 knockout (RAG1(-/-)) mice lacking the adaptive immune system. Healing was investigated using micro-computed tomography (µCT), biomechanical testing, and histologic and mRNA expression analyses. Biomechanical testing demonstrated a significantly higher torsional moment on days 14 and 21 in the RAG1(-/-) mice compared to the WT group. µCT evaluation of RAG1(-/-) specimens showed earlier mineralization and remodeling. Histologically, endochondral ossification and remodeling were accelerated in the RAG1(-/-) compared with the WT mice. Histomorphometric analysis on day 7 showed a significantly higher fraction of bone and a significantly lower fraction of cartilage in the callus of the RAG1(-/-) mice than in the WT mice. Endochondral ossification was accelerated in the RAG1(-/-) mice. Lymphocytes were present during the physiologic repair process, with high numbers in the hematoma on day 3 and during formation of the hard callus on day 14 in the WT mice. Expression of inflammatory cytokines was reduced in the RAG1(-/-) mice. In contrast, expression of anti-inflammatory interleukin 10 (IL-10) was strongly upregulated in RAG1(-/-) mice, indicating protective effects. This study revealed an unexpected phenotype of enhanced fracture healing in RAG1(-/-) mice, suggesting detrimental functions of lymphocytes on fracture healing. The shift from proinflammatory to anti-inflammatory cytokines suggests that immunomodulatory intervention strategies that maximise the regenerative and minimize the destructive effects of inflammation may lead to enhanced fracture repair.

Accelerated fracture healing in mice lacking the 5-lipoxygenase gene.

BACKGROUND AND PURPOSE: Cyclooxygenase-2 (COX-2) promotes inflammation by synthesizing pro-inflammatory prostaglandins from arachidonic acid. Inflammation is an early response to bone fracture, and ablation of COX-2 activity impairs fracture healing. Arachidonic acid is also converted into leukotrienes by 5-lipoxygenase (5-LO). We hypothesized that 5-LO is a negative regulator of fracture healing and that in the absence of COX-2, excess leukotrienes synthesized by 5-LO will impair fracture healing. METHODS: Fracture healing was assessed in mice with a targeted 5-LO mutation (5-LO(KO) mice) and control mice by radiographic and histological observations, and measured by histomorphometry and torsional mechanical testing. To assess effects on arachidonic acid metabolism, prostaglandin E2, F2α, and leukotriene B4 levels were measured in the fracture calluses of control, 5-LO(KO) COX-1(KO), and COX-2(KO) mice by enzyme linked immunoassays. RESULTS: Femur fractures in 5-LO(KO) mice rapidly developed a cartilaginous callus that was replaced with bone to heal fractures faster than in control mice. Femurs from 5-LO(KO) mice had substantially better mechanical properties after 1 month of healing than did control mice. Callus leukotriene levels were 4-fold higher in mice homozygous for a targeted mutation in the COX-2 gene (COX-2(KO)), which indicated that arachidonic acid was shunted into the 5-LO pathway in the absence of COX-2. INTERPRETATION: These experiments show that 5-LO negatively regulates fracture healing and that shunting of arachidonic acid into the 5-LO pathway may account, at least in part, for the impaired fracture healing response observed in COX-2(KO) mice.

Bilateral hand transplantation: bone healing under immunosuppression with tacrolimus, mycophenolate mofetil, and prednisolone.

PURPOSE: Little is known about bone healing after composite tissue transplantation that requires pharmacologic immunosuppression. Bone integration and callus development were assessed in bilateral hand transplantation. METHODS: In this study the course of callus development and callus maturation were assessed by color Doppler sonography and radiography in a double hand transplant and compared with forearm replantation. RESULTS: After hand transplantation, ingrowth of small vessels at the bone junction was observed at week 3, calcified callus became visible at month 4, and bone union was completed at month 11. A similar time course of bone integration was observed after replantation. Plating offered sufficient stability. A recipient periostal flap is thought to have improved blood supply and favored development and induction of callus. CONCLUSIONS: Bone healing after hand transplantation under immunosuppression with tacrolimus, mycophenolate mofetil, and prednisolone is identical to that after forearm replantation.

Oxidant status increased during fracture healing in rats.

We evaluated oxidant status during bone healing in 50 rats. In 40 rats, the right tibia was fractured and fixed intramedullarly (study leg) and the left tibia was pinned but not fractured (control leg). Rats were killed on days 1, 3, 7, 14, 28 and malondialdehyde (MDA) levels were determined in tibial bone tissue. The MDA levels of study and control legs were compared with basal MDA levels in bone in 10 rats. There was no apparent difference between the basal level and control legs, but the study legs showed a statistically significant increase in MDA levels on days 7 and 14. We conclude that no oxidative stress injury occurs during the ischemic period of fracture healing, but it may be significant during inflammation and the formation of callus.

Healing of cortical bone grafts in athymic rats.

We studied healing of allogeneic and syngeneic cortical tibial segment grafts in athymic and normal rats. After 3, 6, and 12 weeks, the weight, circulation, and mineralization rate of the healing segment, and mechanical strength and stiffness of the healing tibia were measured. There were no differences between allogeneic and syngeneic grafts in athymic and normal animals at 3 or 6 weeks. After 12 weeks, the vascularization and mineralization of the grafts, but not of the surrounding callus, were smaller in the allogeneic grafts in the normal recipients than in the other groups. Also after 12 weeks, the stiffness of the healing tibiae was less in allogeneic grafts in normal recipients than in the other groups. The strength of the allogeneic grafts was less than the strength of the syngeneic grafts in both athymic and normal recipients. This suggests that T-cell-mediated rejection is responsible for decreased vascularization and mineralization of allogeneic bone and that the difference in strength between allogeneic and syngeneic grafts is not due to T-lymphocyte graft rejection.

A distinct stratum corneum antigen in psoriasis and its reactions with stratum corneum autoantibodies.

Stratum corneum antibodies are ubiquitous and can be detected by various immunological methods. Of these, the ones detected by hemagglutination undergo changes in antibody titers and have been implicated in psoriasis. The purpose of our study was to examine if differences exist in the activities of the antigens isolated from psoriatic scales in comparison to normal callus. Stratum corneum antigens were prepared by trypsin-phenol-water extraction. The water phase, which contains the stratum corneum antigen, was used to sensitize the red blood cells in the hemagglutination assay. The antibody activity in human sera was determined before and after absorption with antigens isolated from callus, psoriatic scales, and cell envelopes. We found notable differences in the antigens obtained from callus and psoriatic scales. These include higher antibody titers to the antigens of the scales, the presence of unique antigenic determinants on psoriatic scales and the localization of the antigen on cell envelopes. These immunological differences were corroborated by the marked biochemical differences of certain amino acids, most notably glycine and proline, and these differences were unique to psoriatic scales as they were not shared with other hyperproliferative disorders.

An investigation of the contribution of the extraosseous tissues to the diaphyseal fracture callus using a rabbit tibial fracture model and in situ immunocytochemical localisation of osteocalcin.

The extraosseous tissue contribution to diaphyseal fracture callus has been investigated using a rabbit tibial fracture model and osteocalcin immunocytochemistry. The extraosseous tissues were isolated for study by reaming and nailing an osteotomy and excising 2 cm of periosteum on either side of the osteotomy. Specimens obtained from the healing fractures at 1 and 2 weeks after operation, respectively, were decalcified and stained for osteocalcin, a bone-specific protein, using an indirect immunoperoxidase method. The positively stained osteogenic cells appeared to be derived exclusively from the remnant of the periosteum.

Studies in immunodermatology. X. Detection of glycoprotein- and carbohydrate-type stratum corneum antigens by immunofluorescence: conversion effects of enzymes and trauma.

Normal human sera contain stratum corneum (SC) antibodies to two biochemically distinct antigens of normal skin as demonstrated by indirect immunofluorescence (IF) staining with selectively absorbed sera. Absorption of normal human sera with ground callus removed the SC antibodies reactive with the horny layer of normal skin but did not affect the SC antibody titers on cut edges of trypsin-digested callus or lesions of monkey skin induced by scratching. Conversely, absorption of the same sera with a carbohydrate-type SC antigen reduced SC antibody titers on the cut edges of trypsin-digested callus and on lesions of monkey skin induced by scratching but did not alter titers of SC antibodies on normal skin sections.