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In recent years, the incidence of tibial plateau fractures (TPF) has been on the rise. Deep vein thrombosis (DVT) may lead to poor prognosis in patients. The systemic immune-inflammation index(SII) are novel biomarkers of inflammation, and this study aims to verify their predictive effect and construct the nomogram model. This study used binary logistic regression analysis to predict the predictive effect of SII on the occurrence of DVT in tibial plateau fracture patients. And use R studio to construct nomogram model. The results showed that Age (1.03 [1, 1.06], p = 0.032), SII (3.57 [1.68, 7.61], p = 0.04), and NC (7.22 [3.21, 16.26], p < 0.001) were independent predictive factors for DVT. The nomogram demonstrated good predictive performance with small errors in both the training and validation groups, and most clinical patients could benefit from them. The nomogram constructed based on SII can assist clinicians in early assessment of the probability of DVT occurrence., (© 2024. The Author(s).)

Background: Complex regional pain syndrome (CRPS) is a highly disabling cause of pain often precipitated by surgery or trauma to a limb. Both innate and adaptive immunological changes contribute to this syndrome. Dimethyl fumarate (DMF) works through the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor and other targets to activate antioxidant systems and to suppress immune system activation. We hypothesized that DMF would reduce nociceptive, functional, and immunological changes measured in a model of CRPS., Methods: Male C57BL/6 mice were used in the well-characterized tibial fracture model of CRPS. Some groups of mice received DMF 25 mg/kg/d orally, per os for 3 weeks after fracture versus vehicle alone. Homozygous Nrf2 null mutant mice were used as test subjects to address the need for this transcription factor for DMF activity. Allodynia was assessed using von Frey filaments and hindlimb weight-bearing data were collected. The markers of oxidative stress malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were quantified in the skin of the fractured mice using immunoassays along with the innate immune system cytokines IL-1β and IL-6. The accumulation of IgM in the fractured limbs and lymph node hypertrophy were used as indexes of adaptive immune system activation, and the passive transfer of serum from wildtype fractured mice to B cell-deficient fractured muMT mice (mice lacking B cells and immunoglobulin) helped to assess the pronociceptive activity of humoral factors., Results: We observed that oral DMF administration strongly prevented nociceptive sensitization and reduced uneven hindlimb weight bearing after fracture. DMF was also very effective in reducing the accumulation of markers of oxidative stress, activation of innate immune mediator production, lymph node hypertrophy, and the accumulation of IgM in fractured limbs. The sera of fractured vehicle-treated but not DMF-treated mice conferred pronociceptive activity to recipient mice. Unexpectedly, the effects of DMF were largely unchanged in the Nrf2 null mutant mice., Conclusions: Oxidative stress and immune system activation are robust after hindlimb fracture in mice. DMF strongly reduces activation of those systems, and the Nrf2 transcription factor is not required. DMF or drugs working through similar mechanisms might provide effective therapy for CRPS or other conditions where oxidative stress causes immune system activation., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 International Anesthesia Research Society.)

Many diseases display unequal prevalence between sexes. The sex-specific immune response to both injury and persistent pain remains underexplored and would inform treatment paradigms. We utilized high-dimensional mass cytometry to perform a comprehensive analysis of phenotypic and functional immune system differences between male and female mice after orthopedic injury. Multivariate modeling of innate and adaptive immune cell responses after injury using an elastic net algorithm, a regularized regression method, revealed sex-specific divergence at 12 h and 7 days after injury with a stronger immune response to injury in females. At 12 h, females upregulated STAT3 signaling in neutrophils but downregulated STAT1 and STAT6 signals in T regulatory cells, suggesting a lack of engagement of immune suppression pathways by females. Furthermore, at 7 days females upregulated MAPK pathways (p38, ERK, NFkB) in CD4T memory cells, setting up a possible heightened immune memory of painful injury. Taken together, our findings provide the first comprehensive and functional analysis of sex-differences in the immune response to painful injury., (Copyright © 2020 Tawfik, Huck, Baca, Ganio, Haight, Culos, Ghaemi, Phongpreecha, Angst, Clark, Aghaeepour and Gaudilliere.)

Objective: To determine the relationship between inflammation/immune-based indexes and deep venous thrombosis (DVT) incidence rate following tibial plateau fractures METHODS: Retrospective analysis of a prospectively collected data on patients undergoing surgeries of tibial plateau fractures between October 2014 and December 2018 was performed. Duplex ultrasonography (DUS) was routinely used to screen for preoperative DVT of bilateral lower extremities. Data on biomarkers (neutrophil, lymphocyte, monocyte, and platelet counts) at admission were collected, based on which neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), monocyte/lymphocyte (MLR), and systemic immune-inflammation index (SII, neutrophil* platelet/lymphocyte) were calculated. Receiver operating characteristic (ROC) was used to determine the optimal cutoff value for each variable. Multivariate logistic regression analysis was used to evaluate the independent relationship of each biomarker or index with DVT, after adjustment for demographics, co-morbidities, and injury-related variables., Results: Among 1179 patients included, 16.3% (192/1179) of them had a preoperative DVT. Four factors were identified to be significantly associated with DVT, including open fracture, increased D-dimer level. Among the biomarkers and indexes, only platelet and neutrophil were identified to be independently associated with DVT, and the significance remained after exclusion of open fracture. The other independent variables were elevated D-dimer level (> 0.55 mg/L), male gender, and hypertension in the sensitivity analysis with open fractures excluded., Conclusion: These identified factors are conducive to the initial screening for patients at risk of DVT, individualized risk assessment, risk stratification, and accordingly, development of targeted prevention programs.

The aim of this study was to explore the effect of adenosine A2A receptor agonists on fracture healing and the regulation of the immunity system after bone fracture. We implanted fibrin gel containing adenosine A2A receptor agonist CGS 21680/inhibitor ZM 241385/saline locally in rat tibial fracture models, finding that the adenosine A2A receptor agonist could promote fracture healing. At the same time, the adenosine A2A receptor agonist decreased the level of IL-6 in blood and the fracture area, increased Treg cells, and decreased Th17 cells in blood of bone fracture rats. Further, tibial fracture rats implanted with the adenosine A2A receptor agonist gel were injected with IL-6. We found that IL-6 could reverse the effect of adenosine A2A receptor agonists on fracture healing and Treg/Th17 cells in blood. Through the above results, we believe that the adenosine A2A receptor agonist can promote fracture healing and regulate Treg/Th17 cells in blood of rats with fractures. These effects are related to IL-6., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2020 Xi Zheng and Dong Wang.)

Perioperative neurocognitive disorders have been widely recognized as common adverse events after surgical intervention. Aging is one of the most important independent risk factors for worsened cognitive outcome, and this deterioration is linked to exacerbated microglia-mediated neuroinflammation in the aged brain. Under pathological stimulation, microglia are capable of polarizing toward proinflammatory M1 and anti-inflammatory M2 phenotypes. In the present study, we examined how aging affects microglial responses and neuroinflammation following peripheral surgery. Adult (2-3 months) and aged (18 months old) male C57/BL6 mice were subjected to tibial fracture or sham surgery. Aged mice exhibited higher level of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the hippocampus. The expression of synaptic protein synaptophysin (SYP) was also markedly reduced in the aged brain after the surgery. Both adult and aged mice showed significant increases in M1 microglial polarization (CD16/32). In contrast, tibial fracture surgery induced a decreased M2 microglial polarization (CD206, Ym1/2, Arg1) in aged brain but enhanced M2 microglial polarization in adult brain. Aged mice have upregulated voltage-gated proton channel (Hv1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit expression compared with adult mice. The percentage of CD16/32-positive M1 microglia colabeling with Hv1 was higher in aged mice after tibial fracture surgery. Thus, Hv1/NADPH oxidase upregulation in the aged brain may shift the dynamic equilibrium of microglial activation toward M1 polarization and exaggerate postoperative neuroinflammatory responses after peripheral surgical intervention., (© 2019 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Previously, distinct sex differences were observed in the pronociceptive role of spinal immune cells in neuropathic and inflammatory mouse pain models. Both peripheral and central innate and adaptive immune changes contribute to sensitization in the tibia fracture rodent model of complex regional pain syndrome, and the current study evaluated sex differences in the development of pronociceptive immune responses after fracture. At 4 and 7 weeks after fracture, the analgesic effects of a microglia inhibitor were tested in male and female mice, and polymerase chain reaction was used to measure inflammatory mediator expression in skin and spinal cord. The temporal progression of complex regional pain syndrome-like changes in male and female wild-type and muMT fracture mice lacking B cells and antibodies were evaluated, and IgM antibody deposition measured. Pronociceptive effects of injecting wild-type fracture mouse serum into muMT fracture mice were also tested in both sexes, and the role of sex hormones was evaluated in the postfracture development of pronociceptive immune responses. Long-lasting immune changes developed in the fracture limb and corresponding spinal cord of both male and female mice, including upregulated neuropeptide and cytokine signaling, microglial activation, and pronociceptive autoimmunity. These complex postfracture immune responses were sexually dichotomous and interacted in temporally evolving patterns that generated post-traumatic nociceptive sensitization in both sexes lasting for up to 5 months. Unfortunately, the redundancy and plasticity of these chronic post-traumatic immune responses suggest that clinical interventions focusing on any single specific pronociceptive immune change are likely to be ineffectual.

Delayed union and nonunion occur in a minor subject of bone fractures, presenting ongoing challenges to treatment. RANKL, which promotes the differentiation of bone-resorbing osteoclasts, is thought to negatively impact bone healing. In this study, we recruited patients with isolated closed tibial fracture, who were later categorized into normal healing and delayed healing groups based on their healing progression. The regulatory T cell (Treg) compartment was then investigated in each patient. Based on CD45RA and CD62L expression, we distinguished circulating Treg cells into CD45RA + CD62L + naive (N), CD45RA - CD62L + central memory (CM), and CD45RA - CD62L - effector memory (EM) subsets. Compared to normal patients, delayed patients presented significantly lower EM Treg proportion and significantly higher N Treg proportion. Among the N, EM, and CM Treg cells, the EM Treg cells were the most potent at suppressing RANKL expression in T conventional (Tconv) cells. This functionality of EM Treg cells was present in both normal healing patients and delayed healing patients, and was dependent on IL-10, as neutralization of IL-10 resulted in significantly elevated RANKL expression. EM Treg cells presented the highest IL-10 and TGF-β expression directly ex vivo, as well as after anti-CD3/anti-CD28/IL-2 stimulation. CM Treg cells did not present high expression of inhibitory cytokines ex vivo, but was capable of upregulating cytokine expression upon stimulation. N Treg cells, on the other hand, presented limited capacity to upregulate inhibitory cytokines. In summary, our study identified that, while the EM Treg cells were the most effective at suppressing RANKL, they in delayed union patients were present at lower frequencies with functional impairment, resulting in decreased RANKL suppression. Hence, bone-resorbing osteoclast formation may be favored in these patients thus suggesting a possible mechanism for delayed bone healing., (Copyright © 2019 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background: Early diagnosis of acute posttraumatic osteomyelitis (POM) is of vital importance for avoiding devastating complications. Diagnosing POM is difficult due to the lack of a highly specific and sensitive test, such as in myocardial infarct, stroke and intracranial bleeding. Serum inflammatory markers, C-reactive protein (CRP), procalcitonin (PCT), white blood cells (WBC) can support clinical findings but they are not able to differentiate between inflammatory response to infection and the host response to non-infection insult with high specificity and sensitivity., Aim: The objectives of the study were to investigate whether the biochemical and immunoinflammatory patient profile could facilitate postoperative monitoring, guide the antibiotic treatment and timing of revision surgery., Patients and Methods: This prospective nonrandomised cohort study included 86 patients after high-energy injury to the shin requiring primary surgical treatment (open or closed reduction and internal fixation of tibial fracture). Values of the biochemical and immunoinflammatory profile were measured on admission (ADD), first postoperative day (POD1) and fourth-postoperative day (POD4)., Results: We discovered on our sample that the development of POM is associated with increased CRP on ADD, POD1 and decreased albumins on POD4. Further studies are needed to prove that these differences can be useful in diagnosing the risk of infection. The assessment of other important risk factors such as: the extent of soft tissue damage, multiple fractures, transfusion rate, need for conversion primary external fixation to intramedullary (IM) nailing or locking plate fixation can empower our clinical judgment of POM., Conclusions: We can improve prediction of posttraumatic osteomyelitis by using the perioperative inflammatory biomarker CRP in combination with postoperative albumins levels and other associated independent risk factors., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Intramedullary nailing, as the gold standard stabilisation method of most long bones, has been tailed by its extensive use as the basic tool of investigating the immune response to trauma in many large and small animal models, as well as at the clinical setting. Over the last few decades a complex map of interactions between pro and anti-inflammatory pathways has been the result of these significant global research efforts. Parallel to the evolution of modern nailing and reaming techniques, significant developments at the fields of other disciplines relevant to trauma care, has improved the contemporary management of injured patients, challenging previous concepts and altering clinical barriers. The current article aims to summarise the current understanding of the effect of instrumenting the medullary canal after trauma, and hint on potential future directions., (Copyright © 2017. Published by Elsevier Ltd.)

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.

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., Competing Interests: The authors have no conflict of interest. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

The process of bone regeneration after fracture is a complex and well-orchestrated process usually requiring 3-12 weeks. A subset of patients, however, exhibit delayed healing time and even incomplete restoration of the normal bone structure. Although the precise mechanism is unknown, studies have shown that smurf1 may play a role during the process. Here, we sought to determine the involvement of the immune system in impaired bone healing. We found that immediately after fracture, the B-cell composition was shifted toward increased frequency of plasmablasts and decreased frequency of naïve B cells, reflecting higher inflammatory status. The percentage of CD19(+) CD24(+) CD38(+) regulatory B cells was also upregulated in response to bone fracture. The production of IL-10, a pivotal cytokine in regulatory B-cell function, was upregulated in all patients. Interestingly, the increase in IL-10 production was only sustained throughout the healing course in normal healing patients but not in delayed healing patients. Rather, delayed healing patients downregulated B-cell IL-10 secretion early and had reduced level of regulatory B-cell activity. Together, these data revealed a role of regulatory B cells in the endogenous bone regeneration process and an alternation in B-cell-mediated regulation in delayed healing patients., (© 2015 APMIS. Published by John Wiley & Sons Ltd.)

Introduction: Inflammation after trauma is thought to be aggravated by intramedullary nailing (IMN) and predisposes to acute respiratory distress syndrome. Polymorphonuclear granulocytes (PMNs) are the main effector cells in this process. However, in patients with a femur fracture, the injury severity was the decisive factor for the PMN phenotype. A tibia fracture is often caused by a more moderate injury and might allow for a window to assess the innate immune response caused by IMN., Methods: A consecutive series of patients with a tibia fracture were included. The innate immune response was measured before and after IMN by plasma interleukin 6, PMN Mac1, and active FcγRII (FcγRII*) expression both before and after fMLF (N-formylmethionyl-leucyl-phenylalanine) stimulation. Furthermore, HLA-DR on monocytes was analyzed., Results: Twenty-five consecutive patients were included. Polymorphonuclear granulocyte fMLF-induced Mac1 and FcγRII* were decreased. In concordance, HLA-DR expression on monocytes was decreased in patients compared with control subjects. Intramedullary nailing was associated with a further decrease of HLA-DR-positive monocytes, whereas no changes in PMN phenotype or plasma interleukin 6 levels were observed., Conclusion: Intramedullary nailing of a tibial fracture did not affect the PMN phenotype. The impact from injury determined the PMN phenotype. In contrast, the monocyte phenotype changed after the additional insult by IMN in patients with an isolated tibial fracture.

Hemorrhagic shock (hS) interacts with the posttraumatic immune response and fracture healing in multiple trauma. Due to the lack of a long-term survival multiple trauma animal models, no standardized analysis of fracture healing referring the impact of multiple trauma on fracture healing was performed. We propose a new long-term survival (21 days) murine multiple trauma model combining hS (microsurgical cannulation of carotid artery, withdrawl of blood and continuously blood pressure measurement), femoral (osteotomy/external fixation) and tibial fracture (3-point bending technique/antegrade nail). The posttraumatic immune response was measured via IL-6, sIL-6R ELISA. The hS was investigated via macrohemodynamics, blood gas analysis, wet-dry lung ration and histologic analysis of the shock organs. We proposed a new murine long-term survival (21 days) multiple trauma model mimicking clinical relevant injury patterns and previously published human posttraumatic immune response. Based on blood gas analysis and histologic analysis of shock organs we characterized and standardized our murine multiple trauma model. Furthermore, we revealed hemorrhagic shock as a causative factor that triggers sIL-6R formation underscoring the fundamental pathophysiologic role of the transsignaling mechanism in multiple trauma., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

An excessive and uncontrolled systemic inflammatory response is associated with organ failure, immunodepression, and increased susceptibility to nosocomial infection following trauma. Interleukin 6 (IL-6) plays a particularly prominent role in the host immune response after trauma with hemorrhage. However, as a result of its pleiotropic functions, the effect of IL-6 in trauma and hemorrhage is still controversial. It remains unclear whether suppression of IL-6 after hemorrhagic shock and trauma will attenuate organ injury and immunosuppression. In this study, C57BL/6 mice were treated with anti-mouse IL-6 monoclonal antibody immediately prior to resuscitation in an experimental model combining hemorrhagic shock and lower-extremity injury. Interleukin 6 levels and signaling were transiently suppressed following administrations of anti-IL-6 monoclonal antibody following hemorrhagic shock and lower-extremity injury. This resulted in reduced lung and liver injury, as well as suppression in the levels of key inflammatory mediators including IL-10, keratinocyte-derived chemokine, monocyte chemoattractant protein 1, and macrophage inhibitory protein 1α at both 6 and 24 h. Furthermore, the shift to TH2 cytokine production and suppressed lymphocyte response were partly prevented. These results demonstrate that IL-6 is not only a biomarker but also an important driver of injury-induced inflammation and immune suppression in mice. Rapid measurement of IL-6 levels in the early phase of postinjury care could be used to guide IL-6-based interventions.

Bone fracture induces moderate inflammatory responses that are regulated by cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LO) for initiating tissue repair and bone formation. Only a handful of non-invasive techniques focus on monitoring acute inflammation of injured bone currently exists. In the current study, we monitored in vivo inflammation levels during the initial 2 weeks of the inflammatory stage after mouse bone fracture utilizing 50 MHz ultrasound. The acquired ultrasonic images were correlated well with histological examinations. After the bone fracture in the tibia, dynamic changes in the soft tissue at the medial-posterior compartment near the fracture site were monitored by ultrasound on the days of 0, 2, 4, 7, and 14. The corresponding echogenicity increased on the 2nd, 4th, and 7th day, and subsequently declined to basal levels after the 14th day. An increase of cell death was identified by the positive staining of deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and was consistent with ultrasound measurements. The increases of both COX-2 and Leukotriene B4 receptor 1 (BLT1, 5-LO-relative receptor), which are regulators for tissue inflammation, in the immunohistochemistry staining revealed their involvement in bone fracture injury. Monitoring the inflammatory response to various non-steroidal anti-inflammatory drugs (NSAIDs) treatments was investigated by treating injured mice with a daily oral intake of aspirin (Asp), indomethacin (IND), and a selective COX-2 inhibitor (SC-236). The Asp treatment significantly reduced fracture-increased echogenicity (hyperechogenicity, p<0.05) in ultrasound images as well as inhibited cell death, and expression of COX-2 and BLT1. In contrast, treatment with IND or SC-236 did not reduce the hyperechogenicity, as confirmed by cell death (TUNEL) and expression levels of COX-2 or BLT1. Taken together, the current study reports the feasibility of a non-invasive ultrasound method capable of monitoring post-fracture tissue inflammation that positively correlates with histological findings. Results of this study also suggest that this approach may be further applied to elucidate the underlying mechanisms of inflammatory processes and to develop therapeutic strategies for facilitating fracture healing., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In article described research the results of the prevalence of the genetic polymorphism of the gene Methylentetrahydrofolatereductase C677T (MTHFR) in 130 patients with pseudarthrosis of long bones and in those with consolidated fractures. The incidence of allele-T among patients with pseudarthrosis was 1.4 times higher than among those with consolidated fractures. Pathological genotype MTHFR 677-TT was associated with the development avital types of pseudarthrosis and increase the proportion of people with hyperhomocysteinemia, high content of inflammatory mediators and development refracture.

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-1β in the synovial tissue and lower protein levels of IL-1α and IL-1β 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., (Copyright © 2013 by the American College of Rheumatology.)

Bone fracture leads to a cycle of inflammation, cellular migration, and proliferation to restore tissue integrity. Immune cells at the site of injury are involved especially in the early phase of the healing process, but little is known about the cells present in the initial fracture hematoma. The hypothesis of this study was that the cellular composition in a fracture hematoma differs from that found in a muscle hematoma and that these divergences get more pronounced over time. By using a reproducible osteotomy model and muscle trauma in sheep the distributions of the immune cell subpopulations were evaluated 1 and 4 h after surgery. The cell amount within the first 4 h increased in both hematoma. The number of dead cells was higher in the muscle hematoma. One hour postoperatively the initial fracture hematoma revealed a lower granulocyte percentage compared to the muscle hematoma. The ratio of T helper to cytotoxic T cells was higher in the fracture hematoma compared to the muscle hematoma at both investigated time points. B cell percentage increased in the fracture but not in the muscle hematoma from 1 to 4 h. This is the first study that compares the immune cell subpopulations of a fracture and muscle hematoma., ((c) 2009 Orthopaedic Research Society.)

Tibia fracture in rats evokes chronic hindpaw warmth, edema, allodynia, and regional osteopenia resembling the clinical characteristics of patients with complex regional pain syndrome type I (CRPS I). Nerve growth factor (NGF) has been shown to support nociceptive and other types of changes found in neuropathic pain models. We hypothesized that anti-NGF antibodies might reduce one or more of the CRPS I-like features of the rat fracture model. For our studies one distal tibia of each experimental rat was fractured and casted for 4 weeks. The rats were injected with anti-NGF or vehicle at days 17 and 24 post-fracture. Nociceptive testing as well as assessment of edema and hindpaw warmth were followed during this period. Molecular and biochemical techniques were used to follow cytokine, NGF and neuropeptide levels in hindpaw skin and sciatic nerves. Lumbar spinal cord Fos immunostaining was performed. Bone microarchitecture was measured using microcomputed tomography (microCT). We found that tibia fracture upregulated NGF expression in hindpaw skin and tibia bone along with sciatic nerve neuropeptide content. We also found nociceptive sensitization, enhanced spinal cord Fos expression, osteopenia and enhanced cytokine content of hindpaw skin on the side of the fracture. Anti-NGF treatment reduced neuropeptide levels in sciatic nerve and reduced nociceptive sensitization. There was less spinal cord Fos expression and bone loss in the anti-NGF treated animals. Conversely, anti-NGF did not decrease hindpaw edema, warmth or cytokine production. Collectively, anti-NGF reduced some but not all signs characteristic of CRPS illustrating the complexity of CRPS pathogenesis and NGF signaling.

Background: Damage of tissues by mechanical injury and inflammation is followed by reaction of the regional lymphoid tissue, lymphatics, and lymph nodes. In our previous lymphoscintigraphic studies, we showed that closed fractures of a lower limb cause reaction of the local lymphoid tissue. There was dilation of lymphatics draining the site of the fracture and enlargement of inguinal lymph nodes. These changes persisted even after clinical healing of the fracture. In the long-lasting nonhealing fractures, the lymphoscintigraphic pictures were different. The draining lymphatics became obliterated, and the lymph nodes disappeared., Methods: In this study, we tried to correlate the lymphoscintigraphic images, reflecting the immune events at the fracture site, with the immunohistochemical observations of the biopsy specimens obtained during corrective operations from the healing and nonhealing fracture gaps. Thirty-eight patients with closed fracture of the tibia without traumatic skin changes were studied., Results: We confirmed that closed tibial fracture evokes response of the regional lymphatic system. Normal fracture healing with immune cell infiltrates and foci of ossification was accompanied by dilated lymphatics and enlarged lymph nodes. Prolonged nonhealing fracture with lack of cellular reaction in the gap proceeded with decreased mass of lymph nodes., Conclusion: This study provides evidence for existence of a functional axis between wound of bone and surrounding soft tissue and the local lymphatic (immune) system. We hypothesize that the fast healing is regulated by influx into the wound of lymph node regulatory cells, whereas prolonged healing causes gradual exhaustion of the regional lymph node functional elements, and reciprocally impairment in sending regulatory cells to the fracture gap.

Surgery and accidental trauma induce changes in the immune response, showing a predominant pattern of activation through the Th2 cell pathway to the detriment of Th1 cell pathway activation. Anapsos is a hydrosoluble extract obtained from Polypodium leucotomos. Anapsos has shown immunomodulating effects in vitro. On a rat experimental model (tibia and fibula fracture), cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, and IL-12) (enzyme-linked immunosorbent assay, ELISA) and cell percentages of CD4, CD8 CD25, CD122, and CD132 (monoclonal antibodies, MoAb) were determined in peripheral blood 7 days before surgery (PRE), 1 day after surgery (1PO), and 7 days after surgery (7PO). On postoperative day 1, rats undergoing fracture show an increase of CD8 percent expression and IL-6 and IL-10 levels, in contrast to rats undergoing fracture plus anapsos treatment. On postoperative day 7, rats undergoing fracture show an increase of IL-6 levels, whereas rats undergoing fracture plus anapsos do not. The IL-12 level decreases on postoperative day 7 in the group with fracture but not in the fracture plus anapsos group. Thus, we conclude that anapsos is able to modulate the immune response after trauma, inhibiting Th2 pathway activation with no effect on Th1 pathway activation. In trauma, Anapsos could prevent the shifting Th1/Th2 balance.

Objective and Design: Cytokine-mediated immunoresponses are consequences of isolated traumatic brain injury (TBI) and muskuloskeletal trauma but little is known when both impacts occur simulanteously in combined neurotrauma (CNT), i. e. TBI + muskuloskeletal trauma (bone fracture)., Materials and Methods: A "two-hit"-experimental model of CNT (TBI + tibia fracture) was used to investigate circulating cytokine interleukin-1-beta, -6, -10 and sTNF-R1 concentrations following peripheral bone fracture only, TBI only and CNT. Blood samples were drawn at 30 min, 6 h, 24 h, 48 h, and 7 days following trauma and circulating cytokine concentrations were determined via immunoassay., Results: Circulating cytokines were increased after trauma (p <0.001 vs. controls), but peaked at different time points. sTNF R1 peaked first at 30 min, followed by IL-6 at 6 h after trauma. IL-10 levels were highest at 24 h, and those for IL-1beta at 48 h after trauma. Circulating IL-6 and IL-10 levels were highest in CNT at 8/10 time points studied (p <0.001)., Conclusion: Circulating cytokine IL-1-beta, -6, -10 and sTNF-R1 concentrations are increased after trauma (TBI, fracture and CNT) but peak at different time points. Pronounced IL-6 and IL-10 responses after CNT may contribute to the increased susceptibility for complications in CNT versus monotrauma.

Critical size defects of bone and delayed fracture healing due to metabolic disorders are still problems in orthopaedic surgery. Adenoviral vectors encoding bone morphogenetic protein-2 (Ad.BMP-2) have been used to stimulate bone formation in small animals. The present study evaluated the use of direct adenoviral gene transfer for inducing bone formation in a large animal. Standardized iliac crest defects were created surgically on both sides of the pelvic bone of white mountain sheep. The efficiency of gene transfer was evaluated using recombinant adenoviruses carrying the cDNA for luciferase. High levels of transgene expression, restricted to the site of injection, were found for the 1st week. Transgene expression then fell considerably, but could still be detected for up to 5 weeks. To investigate the effect on bone healing, Ad.BMP-2 (10(11) particles in 200 mul saline) was unilaterally injected into iliac crest defects and into tibial osteotomies. The contralateral defects remained untreated to evaluate possible systemic effects. The controls were treated with saline solution. Bone formation within the defect, assessed by micro-computed tomography (CT) measurement at 8 weeks, and callus formation after osteotomy were significantly reduced following direct application of Ad.BMP-2. The retardation compared to untreated control animals was additionally found at the contralateral iliac crest indicating a systemic inhibitory effect. Histological analysis confirmed the CT measurement and showed an increased number of inflammatory cells within both defects. Antibodies against the adenovirus and the transgene product were detected in all treated animals. These data show a systemic retardation of bone formation following a single local injection of Ad.BMP-2 in sheep. This finding stands in contrast to the data obtained from small animal models. Further studies are needed to determine the contribution of the immune response to these results, and whether a lower dose of Ad.BMP-2 would be advantageous.

Background: Elevated plasma elastase levels have been reported following major trauma and isolated femoral fracture. Reamed femoral nailing has been shown to further increase plasma elastase levels. The aim of this study was to investigate polymorphonuclear neutrophil (PMN) priming for degranulation following major trauma and isolated long-bone/pelvis fracture by assessing the ability of PMN to release elastase in vitro in response to a stimulus., Methods: We further analyzed PMN surface expression of the integrins CD11b and CD18 as markers of PMN activation. Ten major trauma (Injury Severity Score>or=18) patients and 12 patients with isolated long-bone/pelvis fracture were included in the study. Patients in the isolated fracture group were further stratified into reamed nail and external-fixation groups following surgery., Results: A significant increase in the capacity of PMN to release elastase was seen following major trauma, but not in isolated fracture patients. Surgery did not further alter PMN elastase release. CD11b and CD18 expression was essentially unaltered in all groups., Conclusions: PMN is primed for increased degranulation following major trauma but not following isolated long-bone/pelvis fracture. Accumulation of primed, hyperactive PMN into tissues can lead to severe tissue damage and thus multiple organ failure.

Mechanical conditions at the fracture line determine the mode of fracture healing (osteonal versus non-osteonal bone union). The aim of this study was to investigate the influence of differing degrees of fracture stability on the time course of chondrogenesis, enchondral ossification and immigration of macrophages into the fracture callus. Using a fracture model of the rat's tibia, histological (Azan staining), immunohistological (antibodies directed against the macrophage-specific surface antigen ED2), and molecular biological techniques (expression of the mRNA of the cartilage-specific collagen IX, osteocalcin - a marker for mature osteoblasts - and the macrophage-specific macrosialin) were employed. In terms of histology and molecular biology (collagen IX mRNA expression) chondrogenesis in the fracture gap continued for longer in less stable fractures. In more stable fractures bone formation - identified by osteocalcin mRNA expression - increased from day 12 onwards. The expression of the macrophage-specific surface antigen ED2 and the mRNA of macrosialin was more pronounced but of shorter duration in the more stable fractures. This study shows that differing degrees of fracture stability not only influence the interplay between osteogenesis and chondrogenesis but also alter the kinetics of macrophage immigration into the fracture callus. These findings could aid in better understanding the cytobiologic mechanisms of callus formation and may suggest that macrophages are an important factor not only in soft tissue healing but also in bone healing.

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.

We studied the effects of the immunosuppressant cyclosporin A on bone ingrowth into allo- and xenografts in the rat using titanium bone chambers. The bone chambers were implanted bilaterally in the tibia. Each rat had one allograft and one xenograft. At 6 weeks the distance of soft tissue and bone ingrowth was measured on histological slides. In xenografts, soft-tissue ingrowth was only slightly less than in allografts, but the ingrown tissue became much less ossified. These differences were unaffected by cyclosporin A treatment, suggesting that the lesser amount of bone formation in xenografts is only weakly associated with T-cell-dependent immune reactions.

Background: Natural killer cells (NKCs) participate in "innate" cell-mediated immunity. Fracture/soft tissue injuries are cytokine rich and may influence cell-mediated immunity., Objective: To study the effects of fracture cytokines on NKC function., Design: A case-control study., Setting: A level I trauma center and laboratory in a university medical center., Participants: Patients requiring open fracture fixation and healthy volunteers., Interventions: Fracture supernatants and peripheral plasma were collected during open fracture fixation. Volunteer mononuclear cells were used as effector (NKC) sources. Mononuclear cells were preincubated with fracture supernatants, paired peripheral plasma, or normal plasma under various conditions., Main Outcome Measures: Natural killer cell lysis of K562 target cells was assessed by chromium 51 release., Results: Fracture supernatants suppressed NKC function more rapidly than peripheral plasma. Fracture supernatants from 1 to 4 days after injury were most suppressive. Inactivation of complement and reactive oxygen species failed to restore lysis. Neutralizing antibodies to interleukin 4 and interleukin 10 further suppressed lysis. Antibodies to transforming growth factor beta1 failed to restore lysis. The addition of interferon gamma did not restore lysis but the addition of interleukin 12 did., Conclusions: Fracture supernatants and peripheral plasma from patients with fractures suppress NKCs. The responsible mediators may be concentrated in fracture/soft tissue injuries. Responses to manipulation of the cytokine environment suggest that fracture cytokines may impair cooperation between NKCs and accessory cells.

Fourteen patients with 15 open tibial fractures were examined retrospectively to compare postoperative infection rates of asymptomatic patients who tested positive or negative for the human immunodeficiency virus antibody (HIVab). All patients were treated with a standard open-fracture protocol. All of the HIVab-positive patients developed postoperative infections. There were five postoperative infections in 4 patients; 3 of the 4 patients were HIVab-positive and 2 of these patients developed chronic osteomyelitis. The only other infection, in an HIVab-negative patient with insulin-dependent diabetes, resolved without complication. All other HIVab-negative patients had no complications. The infection rate for asymptomatic HIVab-positive patients with open tibial fractures was higher than that for HIVab-negative patients in our study. These data suggest that the HIV status of patients with open tibial fractures is relevant to treatment outcome.

We studied tissue samples of noninfected delayed union or nonunion of diaphyseal bones in 10 patients immunopathologically and neuroimmunologically 4 to 25 months after the primary injury. Samples mostly consisted of vascularized connective tissue of varying density with the proline-4-hydroxylase-containing fibroblast as the major cell type. Most inflammatory cells were CD4 T-lymphocytes and their number was always twice that of the CD8 positive cells. Staining for CD11b positive monocyte/macrophages showed in all samples positive cells scattered in the connective tissue stroma with perivascular enrichments. Mast cells were absent or very rare. Our findings suggest that delayed union and nonunion tissue consists of vascularized connective tissue, which mostly contains 5B5 fibroblasts, CD11b macrophages and vascular endothelial cells with only few immigrant recently recruited monocytes or lymphoid cells. Almost all resident cells seem to be involved in tissue remodeling as suggested by their content of fibroblast-type MMP-1 and its proteolytic activator MMP-3 or stromelysin. The most striking finding was the paucity or total lack of peripheral innervation, which may have to do with the nonunion of the fracture.

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.

The present investigation shows that chronic posttraumatic osteitis is associated with markedly elevated prostaglandin E2 (PGE2) serum levels. Synthesis of PGE2 is inhibited by nonsteroidal antiinflammatory drugs, such as ibuprofen, which block cyclooxygenase. Osteitis patients showed significantly decreased PGE2 serum levels during treatment with ibuprofen (1200 mg/day). In addition, a significant increase in soluble interleucin-2 receptor serum levels was observed. PGE2 has significant immunosuppressive effects on cell-mediated immunity. On the other hand, soluble interleucin-2 receptor is a reliable indicator of immune system activation. Ibuprofen seems to be a potent immunostimulating agent for the treatment of chronic posttraumatic osteitis, which is characterized by depression of cell-mediated immunity.

The level of immunoglobulins, complement and properdin, data of leukograms were studied in 40 patients with closed fractures of bones with noncomplicated and complicated (by a purulent infection) course of the posttraumatic period. The authors have established variants of changes of these parameters, their correlation with the adaptation reactions of the organism and severity of the trauma.