Human early fracture hematoma is characterized by inflammation and hypoxia.

BACKGROUND: An effective immune system, especially during the inflammatory phase, putatively influences the quality and likelihood of bone healing. If and how this is reflected within the initial fracture hematoma is unclear. QUESTIONS/PURPOSES: We therefore asked the following questions: (1) Does the local expression in fracture hematoma of genes involved in adaptation to hypoxia, migration, angiogenesis, and osteogenesis vary as compared to the peripheral blood? (2) Do these changes occur time dependently? (3) Is the gene expression during fracture hematoma formation altered by irradiation? METHODS: Cells from fracture hematoma of 20 patients and hematomas formed in 40 patients after THA (20 without and 20 with preoperative radiation) were isolated and RNA was extracted to analyze the influence of oxygen deprivation during fracture healing on mRNA expression of genes (HIF1A, LDHA, and PGK1) involved in immunoregulation (IL6, IL8, CXCR4), angiogenesis (VEGF, IL8), and osteogenesis (SPP1, RUNX2) by quantitative PCR. RESULTS: We observed locally increased LDHA gene expression in fracture hematoma cells (6-72 h post fracture) reflecting the adaptation to hypoxia. IL6, IL8, and VEGF upregulation indicated hypoxia-mediated inflammation and angiogenesis; increased CXCR4 expression reflected immigration of immune cells. Osteogenic differentiation was reflected in the increased expression of the SPP1 and RUNX2 genes. The increased expression of the LDHA, VEGF, IL8, SPP1 and RUNX2 genes occurred time dependently. Irradiation suppressed HIF1A, IL6, IL8, CXCR4, and RUNX2 gene expression. CONCLUSIONS: Our data suggest cells in the fracture hematoma (1) adapt to hypoxia and (2) promote inflammation in fracture healing at the mRNA level, indicating early involvement of the immune system. CLINICAL RELEVANCE: The initial fracture hematoma is important for the onset of angiogenesis, chemotaxis, and osteogenesis.

[Effects of Tongmai decoction on the perioperative changes of plasma TNF-alpha and IL-6 in patients with femoral fractures].

OBJECTIVE: To explore the effects of Tongmai decoction on the perioperative changes of serum concentrations of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6 in patients with femoral fractures, and conform the effectiveness of Tongmai decoction on inflammatory factors in patients with femoral fractures, providing the theoretical evidence for the clinical use of Tongmai decoction. METHODS: From October 2007 to May 2009, 60 patients with closed traumatic femoral fractures were selected according to the inclusion criterias and exclusion criterias. All the patients were randomly divided into three groups (group A, group B and group C). Twenty patients in group A (Tpanax Notoginseng pill group), 13 patients were male and 7 patients were female; ranging in age from 20 to 45 years, averaged 32.0 years; the disease course ranged from 2.0 to 26.0 h, with an average of 9.5 h. Twenty patients in group B (Tpanax Notoginseng pills and Lornoxicam injection group),12 patients were male and 8 patients were female; ranging in age from 23 to 42 years, averaged 31.0 years; the disease course ranged from 3.5 to 25.0 h, with an average of 13.6 h. Twenty patients in group C (Tpanax Notoginseng pill, Lornoxicam injection and Tongmai decoction group), 14 patients were male and 6 patients were female; ranging in age from 21 to 44 years, averaged 31.5 years; the disease course ranged from 4.6 to 29.0 h, with an average of 13.3 h. Among all the patients, 42 patients with fractures were fixed with femoral intramedullary nailing, and other 18 patients with femoral locking plate fixation. The patients in group A took Tpanax Notoginseng pills orally, 4 g each time and twice daily; the patients in group B took Tpanax Notoginseng pills orally as group A, and at the same time received intramuscular injection of Lornoxicam, 8 mg each time and once daily; the patients in group C took Tpanax Notoginseng pills orally and received intramuscular injection of Lornoxicam as group B, and at the same time took Tongmai decoction (R ) orally, 200 ml each time and twice daily. The above medications were administered to the three groups on the second day after admission to hospital. Peripheral blood samples were taken for determination of pro-inflammatory cytokines of TNF-alpha and IL-6 in blood serum on the 2nd and 6th days before operation and on the 8th and 13th days after operation. And all the patients were evaluated liver and kidney function at the 2nd and 7th days after admission. Analysis of variance and least significant difference-test were done with the help of SPSS 17.0 statistic software. RESULTS: The differences among three groups of TNF-alpha and IL-6 in blood serum at the 2nd day after admission and 2 days after operation had no statistical significance (P > 0.05). The TNF-alpha and IL-6 levels among 3 groups had statistical differences at the 7th day after admission and at the 7th day after operation (P < 0.05, P < 0.01). There were significant differences of TNF-alpha and IL-6 levels between the 7th day after admission and the 2nd day after admission, the 7th day after operation and the 2nd day after admission (P < 0.01). There were also significant differences of TNF-alpha and IL-6 levels between group C compared with group A and B at the 7th day after admission and the 7th day after operation(P < 0.05, P < 0.01). CONCLUSION: The serum concentrations of TNF-alpha and IL-6 level significantly increased in perioperative period. The results indicate that the Tongmai decoction may play an important role in inhibiting the release of TNF-alpha and IL-6 into the blood stream and decreasing the incunabula complication at early traumatic stage.

[Roles of dendritic cells in mediating decreased delayed type hypersensitivity responses after trauma].

OBJECTIVE: To study the effects of hemorrhage combined with closed fracture on delayed type hypersensitivity (DTH) responses in mice and to explore the relevant mechanisms. METHODS: DTH responses were induced with 2, 4-dinitro-1-fluorobenzene (DNFB) or fluorescein isothiocyanate (FITC) skin painting after injury, and single cell suspensions from pooled inguinal lymph nodes were analyzed by flow cytometry for FITC+ cells and dendritic cells (DC). The ability of cells from pooled inguinal lymph nodes was tested 24 hours after skin painting with DNFB in transferring sensitization for DTH to DNFB. RESULTS: The DTH responses after injury decreased significantly compared with that of sham-injured mice (P<0.01). Flow cytometry showed that FITC+ cells, FITC+/CD11c+ cells, and FITC+/CD11c+ / major histocompatibility complex II+ cells were all significantly decreased after trauma (P<0.01). The ability of cells to transfer sensitization for DTH to DNFB also declined (P<0.01). CONCLUSION: Hemorrhage combined with closed fracture decreases the DTH responses in mice, which may be attributed to the reduced antigen-presenting capacity of DC in the injured mice.

[Research on the expression of some immune markers after reamed and unreamed femoral nailing].

OBJECTIVE: To investigate on the expression of some cytokines and other immunity makers right after the operation, the effect of femoral nailing on systemic immunity and sought to differentiate any differences between reamed and unreamed IMN. METHODS: Fifty-nine patients presenting with acute femoral fractured including 55 male and 4 female, 32.1 years old on average, are divided into 2 group depend on ISS. All patients were treated by close reduction and intramedullary nail for fixation. In group 1, 23 reamed and 23 unreamed; in group 2, 7 reamed and 6 unreamed. Venous blood samples were taken at 24 hr pre-operationally, and 1 hr, 24 hr, 48 hr post operationally. Serum TNF, IL-6, IL-8, IL-10 were measured by enzyme-linked immunosorbent assay. CRP was measured by protein assay apparatus. We also collected venous samples from 22 healthy uninjured volunteers, which formed control group. RESULTS: All immune marks were elevated post operation, for IL-6, IL-8, IL-10, this elevation began at 1 hr after operation, reached to the peak at 24 hr, and then down but never to the normal at 48 hr. For TNF and CRP, the level were raised at 24 hr, and then fallen at 48 hr. All mediators were raised significantly above the control group (< 0.05). Between reamed and unreamed patients both in group 1 and group 2, Although there was a trend towards higher levels of TNF, IL-6, IL-8, IL-10 and CRP in RFN than in the URFN, no significant difference was found except that there was a greater release of serum IL-10 in RFN than in URFN at 24 hr post operation (P = 0.047). Two patients have become SIRS, but the markers have shown no significant difference with those that have no SIRS symptoms. CONCLUSIONS: To the patient not injured severely, using IMN for treatment will make the inflammatory mediators re-released on higher level than normal, which will be balanced by immunity itself soon, so IMN won't make any damage severely. And no significant difference were found between reamed and unreamed nail. But the changing of IL-10 show us that after IMN, especially the reamed nailing, the level of anti-inflammatory mediators will show the difference more apparently between RFN and URFN while the patient got injured more severely. Under this condition, the RFN will aggravate the restrain of immunity.

Biochemical changes after trauma and skeletal surgery of the lower extremity: quantification of the operative burden.

OBJECTIVE: To quantify changes in variables of inflammation, coagulation, and fibrinolysis in blunt trauma patients with lower extremity fractures who underwent different types of surgical procedures. DESIGN: Prospective, cohort study. SETTING: Level I university trauma center. PATIENTS: We allocated 83 blunt trauma patients in stable condition and 22 patients eligible for elective hip replacement to four treatment groups. INTERVENTIONS: In 34 multiply traumatized patients with femoral fracture (group PTFF) and in 28 patients with an isolated femoral fracture (group IFF), primary unreamed intramedullary nailing for stabilization of the femoral shaft fracture was performed. In 22 patients, an elective uncemented total hip arthroplasty (group THA) was inserted for osteoarthritis, and in 21 control patients, an isolated ankle fracture (group AF) was acutely stabilized. MEASUREMENTS AND MAIN RESULTS: From serially sampled central venous blood, the perioperative concentrations of interleukin (IL)-6, of tumor necrosis factor-alpha, of prothrombin fragments 1 + 2, and of D-dimer cross-linked fibrin degradation products were evaluated. Intramedullary instrumentation for an isolated femur fracture caused a significant perioperative increase in the concentrations of IL-6 (preoperative IL-6, 52 +/- 12 pg/mL; IL-6 30 mins postinsertion, 78 +/- 14 pg/mL; p = .02). This increase was comparable with group THA (preoperative IL-6, 46 +/- 16 pg/mL; IL-6 30 mins postinsertion, 67 +/- 11 pg/mL; p = .03). A positive correlation occurred between both groups (r = .83, p < .0004). Multiple trauma patients demonstrated significantly (p = .0002) higher IL-6 concentrations than all other groups throughout the study period and showed a significant increase after femoral nailing (preoperative IL-6, 570 +/- 21 pg/mL; IL-6 30 mins postinsertion, 690 +/- 24 pg/mL; p = .003), whereas no perioperative change was seen in group AF. The highest IL-6 increases were associated with a longer ventilation time (group PTFF) and a longer period of positive fluid balances (groups PTFF, IFF, THA). The coagulatory variables demonstrated similar perioperative increases in groups IFF and THA, but not in groups PTFF and AF. The IL-6 concentrations and the prothrombin fragments 1 + 2 concentrations correlated between groups THA and IFF at 30 mins and at 1 hr after surgery (r2 = .64, p < .02). In all patients the clinical variables were stable perioperatively. CONCLUSIONS: Major surgery of the lower extremity causes changes to the inflammatory, fibrinolytic, and coagulatory cascades in patients with stable cardiopulmonary function. The inflammatory response induced by femoral nailing is biochemically comparable to that induced by uncemented total hip arthroplasty. In multiple trauma patients, increases, which occurred in addition to those induced by the initial trauma, were measured. Definitive primary femoral stabilization by intramedullary nailing imposes an additional burden to the patient with blunt trauma. A careful preoperative investigation is required to evaluate whether primary definitive stabilization can be performed safely.

Severe depression of host immune functions following closed-bone fracture, soft-tissue trauma, and hemorrhagic shock.

OBJECTIVE: To determine the contribution of soft-tissue trauma plus hemorrhage, bone fracture and hemorrhage, as well as the contribution of bone fracture, soft-tissue trauma and hemorrhage on host immune function. SUBJECTS: Adult male mice (n = 6/group). DESIGN: Prospective, randomized, controlled study. SETTING: Animal laboratory at a university-affiliated hospital. INTERVENTIONS: Closed-bone fracture (right lower leg; external fixation) and/or soft-tissue trauma (2.5-cm midline laparotomy, closed in two layers) were induced before hemorrhagic shock (mean arterial blood pressure of 35 +/- 5 (SEM) mm Hg for 90 mins, followed by fluid resuscitation) in male C3H/HeN mice and the animals were killed at 72 hrs after initiation of the experiment. MEASUREMENTS AND MAIN RESULTS: Splenocyte interleukin (IL)-2 and IL-3 release capacity, as well as splenic and peritoneal macrophage IL-1 and IL-6 release capacity were determined. Different traumatic insults, i.e., bone fracture or soft-tissue trauma in conjunction with hemorrhage, produced comparable immune depression. More significant depression of splenocyte IL-2 and IL-3 release capacity as well as macrophage IL-1 and IL-6 release capacity occurred with the combined insult (i.e., bone fracture/soft-tissue injury and hemorrhage) than after bone injury or tissue trauma alone with hemorrhage. CONCLUSIONS: The combination of closed-bone fracture and soft-tissue trauma before hemorrhage leads to even more compromised immunity than either soft-tissue trauma or closed-bone fracture along with hemorrhage. The markedly depressed immune function following bone injury, soft-tissue trauma, and hemorrhagic shock may contribute to the increased susceptibility of severely injured patients to sepsis and the ensuing multiple organ failure in the clinical situation.

Suppression of natural killer cell activity in patients with fracture/soft tissue injury.

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.

Immune function is more compromised after closed bone fracture and hemorrhagic shock than hemorrhage alone.

OBJECTIVE: To determine whether closed bone fracture in conjunction with hemorrhagic shock compromises immune functions more severely than hemorrhagic shock alone. DESIGN: In a randomized, controlled trial, closed bone fracture of the right lower leg and/or hemorrhagic shock (mean +/- SEM arterial blood pressure, 35 +/- 5 mm Hg for 90 minutes) were induced in male C3H/HeN mice (weight, 25 g). Animals subjected to hemorrhage were resuscitated with the shed blood and lactated Ringer solution. At 72 hours after the experiment, all animals were killed to obtain whole blood, splenocytes, and splenic and peritoneal macrophages. Macrophage interleukin-1 and splenocyte interleukin-2 and interleukin-3 release were determined by bioassay, and splenocyte proliferation was measured by tritiated thymidine incorporation. RESULTS: Closed bone fracture alone did not affect immune functions 72 hours after the trauma. Hemorrhagic shock, however, induced a significant depression of splenocyte and macrophage functions. Bone fracture followed by hemorrhagic shock further depressed splenocyte proliferation and splenocyte interleukin-2 and interleukin-3 release as well as interleukin-1 release. CONCLUSION: Since bone injury coupled with hemorrhagic shock produces more severe depression of immune functions than hemorrhage alone, bone injury appears to play a contributory role in further depressing immune functions in trauma patients who experience major blood loss.

Activation of Kupffer cells in vivo following femur fracture.

OBJECTIVE: To test the hypothesis that Kupffer cells are activated after blunt femur fracture leading to altered hepatic oxygen (O2) consumption. DESIGN: Prospective randomized experimental trials. SETTING: Laboratory. MATERIALS AND METHODS: Male Sprague-Dawley rats underwent closed femur fracture with associated soft-tissue injury. Control animals received only anesthesia. After 30 minutes and 2 hours, livers were perfused and fixed. Tissue was processed for scanning and transmission electron microscopy. In separate experiments, hepatic O2 consumption was measured in isolated perfused livers 2 and 48 hours after femur fracture using a Clark-type electrode. Oxygen consumption was calculated from the influent-effluent concentration difference, flow rate, and liver weight. RESULTS: In femur-fractured animals, scanning electron microscopy revealed alterations in Kupffer cell surface characteristics, including increases in cell volume and complex foldings and extensions of the plasma membrane. Transmission electron microscopy showed internal vacuolization and dark-staining granule formation. The changes were more pronounced 2 hours after femur fracture. Hepatic O2 consumption increased significantly at both 2 and 48 hours after femur fracture. Morphologic and functional activation of Kupffer cells were not seen in control animals. CONCLUSION: In vivo ultrastructural evidence shows Kupffer cell activation after closed femur fracture. This activation is associated with increased hepatic O2 consumption, which is present at 2 hours and persists 48 hours following injury. The results suggest that Kupffer cell activation may be related to the acute-phase response following trauma.

[A means for determining the bactericidal activity of the tissues during the treatment of orthopedic patients by transosseous osteosynthesis methods]. / Sposob opredeleniia bakteritsidnoi aktivnosti tkanei pri lechenii ortopedicheskikh bol'nykh metodami chreskostnogo osteosinteza.

The author suggests a new method for the assessment of the bactericidal activity of local tissues in the treatment of bone fractures or after effects thereof by the transosseous osteosynthesis methods. Tissue components may induce the formation of large sites of delayed growth in standard Micrococcus lysodeikticus cultures. To assess the bactericidal activity of the tissues, a standard diagnostic disk with an antibiotic is placed onto such cultures, this permitting the expression of the tissue bactericidal activity in equivalents of the antibiotic antibacterial effect and determine the low, medium, and high levels of the tissue bactericidal activity with respect to the minimal therapeutic suppressing concentration of this antibiotic. The author suggests that the mechanism of a drastic elevation of the local tissue bactericidal activity in transosseous osteosynthesis is explained by an essential enhancement of local tissue metabolism.

The role of T-system immunity in reparatory regeneration of the bone tissue in animals.

It has been demonstrated in experiments on mice (CBA X C57BL) X F1, thymectomized and irradiated by 800 R, with the haemopoietic system restored by bone marrow (B-mice) that in these animals, as compared with the controls, the changes in cellular immunity (inhibition of natural killer cells and stimulation of individual functions of the phagocytizing cells) are accompanied by considerable inhibition of osteogenesis. Compensatory regeneration of broken thigh-bone in B-mice is delayed by 5--10 days in various elements of the regenerated tissue in comparison with normal mice. Anatomical formation of the provisional callus in B-mice is not completed on day 21 and approaches a 14-day regenerate of the controls. The obtained results suggest the participation of T-system immunity in the reparatory regeneration of bone tissue.

[Antibodies against staphylococci in patients of a department for orthopedic surgery (author's transl)]. / Staphylokokken-Antikörper-Titer bei unfallchirurgischen Patienten. I. Mitteilung.

Sera from different groups of patients in a department of traumatic surgery were examined for staphylococcal alpha-antitoxin-titres. In comparison to a group of normal controls the following changes were found: 1. Patients, had significantly raised titres, if they had undergone surgery because of closed fractures, pseudarthrosis and luxations and even if the postoperative course was uneventful. This can be explained through the wide-spread occurrence of staphylococci in hospitals, which can invade the organisms through an incision, the skin or mucous membranes. These findings should be kept in mind while estimating staphylococcal antitoxin-titres in patients hospitalized over a long period. 2. Patients suffering from surgical infections and also paraplegics showed still more significantly raised antitoxin-titres in comparison to the normal controls or to patients, who had undergone aseptic surgery. Increased titres were found even in the sera of patients, whose wounds did not drain staphylococcus aureus. Patients suffering from chronic staphylococcal osteomyelitis showed the highest mean values of antitoxin-titres; these patients, however, had also the greatest variations with high and low levels. The question arouse whether a high or a low level corresponds to the severity of the disease. It was found that it is necessary to titrate different antibodies against staphylococcal toxins. In a second paper the results of the simultaneous determination of alpha-antitoxin and anti-PV-leucocidin in patients with posttraumatic bone infections will be presented.