Episodic alcohol exposure attenuates mesenchymal stem cell chondrogenic differentiation during bone fracture callus formation.

BACKGROUND: During bone fracture repair, mesenchymal stem cells (MSC) differentiate into chondrocytes and osteoblasts to form a fracture callus. Our laboratory previously reported that alcohol-exposed rodents with a surgically created tibia fracture display deficient fracture callus formation and diminished signs of endochondral ossification characterized by the absence of chondrocytes and mature hypertrophic chondrocytes, suggesting that alcohol may inhibit MSC differentiation. These findings led to our hypothesis that alcohol exposure inhibits mesenchymal stem cell chondrogenic differentiation within the developing fracture callus. METHODS: In the present study, we utilized a lineage-tracing approach to determine which stage(s) of chondrogenic differentiation are affected by alcohol exposure. We utilized lineage-specific reporter mice to determine the effects of alcohol on MSC and early and late chondrogenic cell frequencies within the fracture callus. In addition, serially sectioned slides were stained immunofluorescently and immunohistochemically and quantified to determine the effect of alcohol on cell proliferation and apoptosis, respectively, within the fracture callus of alcohol-administered rodents. RESULTS: Alcohol-administered rodents had a reduced fracture callus area at 4, 6, and 9 days postfracture. Alcohol had no effect on apoptosis in the fracture callus at any of the examined timepoints. Alcohol-administered rodents had significantly fewer proliferative cells in the fracture callus at 9 days postfracture, but no effect on cell proliferation was observed at earlier fracture callus timepoints. Alcohol-administered rodents had reduced Collagen2a1- and Collagen10a1-expressing cells in the developing fracture callus, suggesting that alcohol inhibits both early chondrogenic differentiation and later chondrocyte maturation during fracture callus development. CONCLUSION: The data suggest that alcohol could affect normal fracture healing through the mitigation of MSC chondrogenic differentiation at the callus site.

Alcohol-induced Wnt signaling inhibition during bone fracture healing is normalized by intermittent parathyroid hormone treatment.

Nearly half of orthopaedic trauma patients are intoxicated at the time of injury, and excess alcohol consumption increases the risk for fracture nonunion. Previous studies show alcohol disrupts fracture associated Wnt signaling required for normal bone fracture repair. Intermittent parathyroid hormone (PTH) promotes bone growth through canonical Wnt signaling, however, no studies have investigated the effect of PTH on alcohol-inhibited bone fracture repair. Male C57BL/6 mice received two-3 day alcohol binges separated by 4 days before receiving a mid-shaft tibia fracture. Postoperatively, mice received PTH daily until euthanasia. Wnt/ß-catenin signaling was analyzed at 9 days post-fracture. As previously observed, acute alcohol exposure resulted in a >2-fold decrease in total and the active form of ß-catenin and a 2-fold increase in inactive ß-catenin within the fracture callus. Intermittent PTH abrogated the effect of alcohol on ß-catenin within the fracture callus. Upstream of ß-catenin, alcohol-treated animals had a 2-fold decrease in total LRP6, the Wnt co-receptor, which was restored with PTH treatment. Alcohol nor PTH had any significant effect on GSK-3ß. These data show that intermittent PTH following a tibia fracture restores normal expression of Wnt signaling proteins within the fracture callus of alcohol-treated mice.

Ethanol Inhibits Mesenchymal Stem Cell Osteochondral Lineage Differentiation Due in Part to an Activation of Forkhead Box Protein O-Specific Signaling.

BACKGROUND: During bone fracture repair, resident mesenchymal stem cells (MSCs) differentiate into chondrocytes, to form a cartilaginous fracture callus, and osteoblasts, to ossify the collagen matrix. Our laboratory previously reported that alcohol administration led to decreased cartilage formation within the fracture callus of rodents and this effect was mitigated by postfracture antioxidant treatment. Forkhead box protein O (FoxO) transcription factors are activated in response to intracellular reactive oxygen species (ROS), and alcohol has been shown to increase ROS. Activation of FoxOs has also been shown to inhibit canonical Wnt signaling, a necessary pathway for MSC differentiation. These findings have led to our hypothesis that alcohol exposure decreases osteochondrogenic differentiation of MSCs through the activation of FoxOs. METHODS: Primary rat MSCs were treated with ethanol (EtOH) and assayed for FoxO expression, FoxO activation, and downstream target expression. Next, MSCs were differentiated toward osteogenic or chondrogenic lineages in the presence of 50 mM EtOH and alterations in osteochondral lineage marker expression were determined. Lastly, osteochondral differentiation experiments were repeated with FoxO1/3 knockdown or with FoxO1/3 inhibitor AS1842856 and osteochondral lineage marker expression was determined. RESULTS: EtOH increased the expression of FoxO3a at mRNA and protein levels in primary cultured MSCs. This was accompanied by an increase in FoxO1 nuclear localization, FoxO1 activation, and downstream catalase expression. Moreover, EtOH exposure decreased expression of osteogenic and chondrogenic lineage markers. FoxO1/3 knockdown restored proosteogenic and prochondrogenic lineage marker expression in the presence of 50 mM EtOH. However, FoxO1/3 inhibitor only restored proosteogenic lineage marker expression. CONCLUSIONS: These data show that EtOH has the ability to inhibit MSC differentiation, and this ability may rely, at least partially, on the activation of FoxO transcription factors.

Impact of Alcohol on Bone Health, Homeostasis and Fracture repair.

PURPOSE OF REVIEW: Alcohol use continues to rise globally. We review the current literature on the effect of alcohol on bone health, homeostasis and fracture repair to highlight what has been learned in people and animal models of alcohol consumption. RECENT FINDINGS: Recently, forkhead box O (FoxO) has been found to be upregulated and activated in mesenchymal stem cells (MSC) exposed to alcohol. FoxO has also been found to modulate Wnt/ß-catenin signaling, which is necessary for MSC differentiation. Recent evidence suggests alcohol activates FoxO signaling, which may be dysregulating Wnt/ß-catenin signaling in MSCs cultured in alcohol. SUMMARY: This review highlights the negative health effects learned from people and chronic and episodic binge alcohol consumption animal models. Studies using chronic alcohol exposure or alcohol exposure then bone fracture repair model have explored several different cellular and molecular signaling pathways important for bone homeostasis and fracture repair, and offer potential for future experiments to explore additional signaling pathways that may be dysregulated by alcohol exposure.

The Effect of Proton Pump Inhibitors on Bone Formation in a Rat Spinal Arthrodesis Model.

STUDY DESIGN: Rat posterolateral arthrodesis model. OBJECTIVE: Quantify the impact of administration of a proton pump inhibitor on spine fusion. SUMMARY OF BACKGROUND DATA: Proton pump inhibitors (PPIs) are widely used for gastrointestinal disorders and for ulcer prophylaxis in patients taking non-steroidal anti-inflammatory drugs. PPIs cause chronic acid suppression which has been found to result in decreased bone mineral density, increased fracture risk, and impaired fracture healing. Despite advances in surgical techniques, pseudarthrosis still occurs in up to 24% of patients requiring revision surgery following spinal fusion procedures. Thus, there are likely many unidentified risk factors. While PPIs have been hypothesized to impact fracture healing, no study has evaluated their effect on spine arthrodesis rates. METHODS: Thirty-eight female rats underwent posterolateral lumbar spinal fusion. Rats were divided into two groups: normal saline control and pantroprazole, which was administered by daily intraperitoneal injections. At 8 weeks postoperative spines were evaluated with manual palpation, microCT, histologic analysis, and biomechanical testing. RESULTS: Fusion rates of the control group and PPI group were not significantly different (100% vs. 94%). Average fusion scores were significantly lower in the pantoprazole group. New bone formation identified on microCT imaging of bilaterally fused specimens demonstrated a lower average volume of newly generated bone in the PPI group, but this difference was not significant. Biomechanical testing demonstrated no significant difference in strength or stiffness of the fusion mass between the groups. CONCLUSION: This study demonstrates that administration of PPIs does not inhibit fusion rates, bone formation, or affect biomechanical integrity of fusion. However, lower fusion scores in the PPI group suggest that a negative impact may still exist. Future studies will explore growth factor and protein expression in the fusion masses as well as utilize higher doses of PPI to fully discern their effect on spine fusion. LEVEL OF EVIDENCE: N/A.

Alcohol exposure decreases osteopontin expression during fracture healing and osteopontin-mediated mesenchymal stem cell migration in vitro.

BACKGROUND: Alcohol consumption is a risk factor for impaired fracture healing, though the mechanism(s) by which this occurs are not well understood. Our laboratory has previously shown that episodic alcohol exposure of rodents negatively affects fracture callus development, callus biomechanics, and cellular signaling which regulates stem cell differentiation. Here, we examine whether alcohol alters chemokine expression and/or signaling activity in the mouse fracture callus during early fracture healing. METHODS: A mouse model for alcohol-impaired tibia fracture healing was utilized. Early fracture callus was examined for alcohol-effects on tissue composition, expression of chemokines involved in MSC migration to the fracture site, and biomechanics. The effects of alcohol on MSC migration and cell adhesion receptors were examined in an in vitro system. RESULTS: Mice exposed to alcohol showed decreased evidence of external callus formation, decreased callus-related osteopontin (OPN) expression levels, and decreased biomechanical stiffness. Alcohol exposure decreased rOPN-mediated MSC migration and integrin ß1 receptor expression in vitro. CONCLUSIONS: The effects of alcohol exposure demonstrated here on fracture callus-associated OPN expression, rOPN-mediated MSC migration in vitro, and MSC integrin ß1 receptor expression in vitro have not been previously reported. Understanding the effects of alcohol exposure on the early stages of fracture repair may allow timely initiation of treatment to mitigate the long-term complications of delayed healing and/or fracture non-union.

Effects of Episodic Alcohol Exposure on BMP2 Signaling During Tibia Fracture Healing.

OBJECTIVES: To explore how alcohol affects the BMP-2 signaling pathway, which is known to play a critical role in bone and cartilage formation during fracture healing. METHODS: A rat model was used to demonstrate the detrimental effects of alcohol exposure on tibia fracture healing. Specific components of the BMP-2 pathway were analyzed in fracture callus on days 3, 7, 14, and 21 after fracture via western immunoassays and enzyme-linked immunosorbent assay. RESULTS: Alcohol exposure before tibia fracture demonstrated attenuation of downstream BMP-2 signaling. The BMP-2 antagonist, Chordin, may be the central component of the BMP-2-related changes demonstrated in this study. Although alcohol affected BMP-related proteins at all time points, it seems that day 14 after fracture is a critical time point for alcohol-related modulation of callus formation in our model. CONCLUSIONS: This study may provide the scientific basis for further studies addressing whether the application of exogenous BMP-2 in patients with a history of alcohol abuse who sustain long bone fractures may or may not be of benefit.

Alcohol-related deficient fracture healing is associated with activation of FoxO transcription factors in mice.

The process of fracture healing is complex, and poor or incomplete healing remains a significant health problem. Proper fracture healing relies upon resident mesenchymal stem cell (MSC) differentiation into chondrocytes and osteoblasts, which are necessary for callus formation and ossification. Alcohol abuse is a leading contributor to poor fracture healing. Although the mechanism behind this action is unknown, excessive alcohol consumption is known to promote systemic oxidative stress. The family of FoxO transcription factors is activated by oxidative stress, and FoxO activation antagonizes Wnt signaling, which regulates mesenchymal stem cell differentiation. We hypothesize that alcohol exposure increases oxidative stress leading to deficient fracture repair by activating FoxO transcription factors within the fracture callus which disrupts chondrogenesis of mesenchymal stem cells. Our laboratory has developed an experimental model of delayed fracture union in mice using ethanol administration. We have found that ethanol administration significantly decreases external, cartilaginous callus formation, and hallmarks of endochondral ossification, and these changes are concomitant with increases in FoxO expression and markers of activation in fracture callus tissue of these mice. We were able to prevent these alcohol-induced effects with the administration of the antioxidant n-acetyl cysteine (NAC), suggesting that alcohol-induced oxidative stress produces the perturbed endochondral ossification and FoxO expression. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2106-2115, 2016.

Alcohol inhibits osteopontin-dependent transforming growth factor-ß1 expression in human mesenchymal stem cells.

Alcohol (EtOH) intoxication is a risk factor for increased morbidity and mortality with traumatic injuries, in part through inhibition of bone fracture healing. Animal models have shown that EtOH decreases fracture callus volume, diameter, and biomechanical strength. Transforming growth factor ß1 (TGF-ß1) and osteopontin (OPN) play important roles in bone remodeling and fracture healing. Mesenchymal stem cells (MSC) reside in bone and are recruited to fracture sites for the healing process. Resident MSC are critical for fracture healing and function as a source of TGF-ß1 induced by local OPN, which acts through the transcription factor myeloid zinc finger 1 (MZF1). The molecular mechanisms responsible for the effect of EtOH on fracture healing are still incompletely understood, and this study investigated the role of EtOH in affecting OPN-dependent TGF-ß1 expression in MSC. We have demonstrated that EtOH inhibits OPN-induced TGF-ß1 protein expression, decreases MZF1-dependent TGF-ß1 transcription and MZF1 transcription, and blocks OPN-induced MZF1 phosphorylation. We also found that PKA signaling enhances OPN-induced TGF-ß1 expression. Last, we showed that EtOH exposure reduces the TGF-ß1 protein levels in mouse fracture callus. We conclude that EtOH acts in a novel mechanism by interfering directly with the OPN-MZF1-TGF-ß1 signaling pathway in MSC.

Exogenous activation of Wnt/ß-catenin signaling attenuates binge alcohol-induced deficient bone fracture healing.

AIMS: Excessive alcohol consumption is associated with fracture non-union. Canonical Wnt pathway signaling activity regulates normal fracture healing. We previously demonstrated that binge alcohol exposure modulates ß-catenin levels in the fracture callus of mice. Here, we sought to determine whether exogenous enhancement ß-catenin signaling activity could restore normal fracture healing to binge-exposed mice. METHODS: C57BL/6 male mice were exposed to episodic alcohol or saline for 6 total days of alcohol exposure over a 2-week period. Following alcohol exposure, mice were subjected to a stabilized mid-shaft tibia fracture. Beginning 4 days post-injury, mice received daily injections of either lithium chloride or saline subcutaneously. Protein levels of activated, inactivated, and total ß-catenin and GSK-3ß in fracture calluses were measured at post-injury day 9. Biomechanical strength testing and histology of callus tissue was assessed at post fracture day 14. RESULTS: Binge alcohol was associated with decreased callus biomechanical strength, and reduced cartilaginous callus formation. Alcohol decreased levels of callus-associated activated ß-catenin while concomitantly increasing the levels of inactive ß-catenin at post-injury day 9. Alcohol also increased callus associated activated GSK-3ß at post-injury day 9. Lithium chloride (an inhibitor of GSK-3ß) treatment increased activated ß-catenin protein levels, significantly decreased activated GSK-3ß and restored cartilaginous callus formation and endochondral ossification. CONCLUSION: These data link alcohol-impaired fracture healing with deregulation of Canonical Wnt signaling activity in the fracture callus. Exogenous activation of the Wnt pathway using LiCl attenuated the damaging effects of binge alcohol exposure on the fracture healing process by modulating canonical Wnt signaling activity.

Ketorolac administration does not delay early fracture healing in a juvenile rat model: a pilot study.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective at controlling pain in children, especially in the treatment of fractures. Adult animal and adult clinical studies demonstrate conflicting evidence for the inhibitory relationship between NSAIDs and fracture healing. Published pediatric orthopaedic clinical studies do not demonstrate an inhibitory effect of ketorolac on bone healing. Little is known about the effects of any NSAID on bone formation in juvenile animals. This study investigates the effects of the NSAID ketorolac on fracture healing in a juvenile rat model. METHODS: Unilateral surgically induced and stabilized tibial shaft fractures were created in 45 juvenile (3 to 4 wk old) male Sprague-Dawley rats. Either ketorolac (5 mg/kg; n=24) or saline (0.9% normal saline; n=21) was then administered to the rats 6 d/wk by intraperitoneal injections. Animals were then randomly assigned into time groups and euthanized at 7 days (n=8 ketorolac, n=7 saline), 14 days (n=8 ketorolac, n=7 saline), or 21 days (n=8 ketorolac, n=7 saline) postfracture. Biomechanical analysis was performed using a custom-designed 4-point bending loading apparatus. Statistics for tibial stiffness and strength data were performed using software package Systat 11. Specimens were also evaluated histologically using hematoxylin and eosin staining. RESULTS: Strength and stiffness of all fractured tibiae increased over time from day 7 to day 21 regardless of treatment type. No statistical difference was found between the fractured tibiae strength or stiffness in the ketorolac or control-treated specimens at the same time point. In addition, the quality of the fracture callus was similar in both groups at each of the time points. CONCLUSIONS: In this study of a juvenile rat model with a stabilized tibia fracture, fracture callus strength, stiffness, and histologic characteristics were not affected by the administration of ketorolac during the first 21 days of fracture healing. CLINICAL RELEVANCE: The absence of inhibitory effects of ketorolac on early juvenile rat fracture healing supports the clinical practice of utilizing NSAIDs for analgesia in children with long bone fractures.

Mesenchymal stem cells facilitate fracture repair in an alcohol-induced impaired healing model.

OBJECTIVES: Clinical studies have shown alcohol to be a risk factor for traumatic orthopaedic injuries and for nonunion. Data from animal studies suggest that alcohol exposure inhibits fracture healing. This report presents a novel rodent model of impaired fracture healing caused by repeated alcohol exposure. Using this model, we examined the regenerative effects of an intravenously administered population of isolated and expanded mesenchymal stem cells (MSCs) on fracture healing. METHODS: Bone marrow-derived MSC were isolated from transgenic green fluorescent protein C57BL/6 mice, and culture expanded using a lineage depletion protocol. Adult wild-type C57BL/6 mice were subjected to a 2-week binge alcohol exposure paradigm (3 days during which they received daily intraperitoneal injections of a 20% alcohol/saline solution followed by a 4-day rest period and another binge cycle for 3 consecutive days). At completion of the second binge cycle, mice were subjected to a mid-shaft tibia fracture while intoxicated. Twenty-four hours after the fracture, animals were administered an intravenous transplant of green fluorescent protein-labeled MSC. Two weeks after the fracture, animals were euthanized and injured tibiae were collected and subjected to biomechanical, histologic, and microcomputed tomography analysis. RESULTS: Pre-injury binge alcohol exposure resulted in a significant impairment in biomechanical strength and decrease in callus volume. MSC transplants restored both fracture callus volume (P < 0.05) and biomechanical strength (P < 0.05) in animals with alcohol-impaired healing. In vivo imaging demonstrated a time-dependent MSC migration to the fracture site. CONCLUSIONS: These data suggest that a 2-week binge alcohol exposure significantly impairs fracture healing in a murine tibia fracture model. Intravenously administered MSC were capable of specifically homing to the fracture site and of normalizing biomechanical, histologic, and microcomputed tomography parameters of healing in animals exposed to alcohol. Understanding MSC recruitment patterns and functional contributions to fracture repair may lead to their use in patients with impaired fracture healing and nonunion.

The biomechanical and histologic effects of platelet-rich plasma on rat rotator cuff repairs.

BACKGROUND: Rotator cuff tears are common injuries that are often treated with surgical repair. Because of the high concentration of growth factors within platelets, platelet-rich plasma (PRP) has the potential to enhance healing in rotator cuff repairs. HYPOTHESIS: Platelet-rich plasma would alter the biomechanical and histologic properties of rotator cuff repair during an acute injury response. STUDY DESIGN: Controlled laboratory study. METHODS: Platelet-rich plasma was produced from inbred donor rats. A tendon-from-bone supraspinatus tear was created surgically and an immediate transosseous repair performed. The control group underwent repair only. The PRP group underwent a repair with PRP augmentation. Rats in each group were sacrificed at 7, 14, and 21 days. The surgically repaired tendons underwent biomechanical testing, including failure load, stiffness, failure strain, and stress relaxation characteristics. Histological analysis evaluated the cellular characteristics of the repair tissue. RESULTS: At 7- and 21-day periods, augmentation with PRP showed statistically significant effects on the biomechanical properties of the repaired rat supraspinatus tear, but failure load was not increased at the 7-, 14-, or 21-day periods (P = .688, .209, and .477, respectively). The control group had significantly higher stiffness at 21 days (P = .006). The control group had higher failure strain at 7 days (P = .02), whereas the PRP group had higher failure strain at 21 days (P = .008). Histologically, the PRP group showed increased fibroblastic response and vascular proliferation at each time point. At 21 days, the collagen fibers in the PRP group were oriented in a more linear fashion toward the tendon footprint. CONCLUSION: In this controlled, rat model study, PRP altered the tissue properties of the supraspinatus tendon without affecting the construct's failure load. CLINICAL RELEVANCE: The decreased tendon tissue stiffness acutely and failure to enhance tendon-to-bone healing of repairs should be considered before augmenting rotator cuff repairs with PRP. Further studies will be necessary to determine the role of PRP in clinical practice.

Acute alcohol exposure impairs fracture healing and deregulates ß-catenin signaling in the fracture callus.

BACKGROUND: Alcohol abuse is a risk factor for bone damage and fracture-related complications. Through precise ß-catenin signaling, canonical Wnt signaling plays a key role in fracture repair by promoting the differentiation of new bone and cartilage cells. In this study, we examined the effects of alcohol on the Wnt pathway in injured bone using a murine model of alcohol-induced impaired fracture healing. METHODS: Male C57Bl/6 or T cell factor (TCF)-transgenic mice were administered 3 daily intraperitoneal doses of alcohol or saline. One hour following the final injection, mice were subjected to a stabilized, mid-shaft tibial fracture. Injured and contralateral tibias were harvested at 6, 9, or 14 days post-fracture for the analysis of biomechanical strength, callus tissue composition, and Wnt/ß-catenin signaling. RESULTS: Acute alcohol treatment was associated with a significant decrease in fracture callus volume, diameter, and biomechanical strength at day 14 post-fracture. Histology revealed an alcohol-related reduction in cartilage and bone formation at the fracture site, and that alcohol inhibited normal cartilage maturation. Acute alcohol exposure caused a significant 2.3-fold increase in total ß-catenin protein at day 6 and a significant decrease of 53 and 56% at days 9 and 14, respectively. lacZ staining in ß-galactosidase-expressing TCF-transgenic mice revealed spatial and quantitative differences in Wnt-specific transcriptional activation at day 6 in the alcohol group. Days 9 and 14 post-fracture showed that acute alcohol exposure decreased Wnt transcriptional activation, which correlates with the modulation of total ß-catenin protein levels observed at these time points. CONCLUSIONS: Acute alcohol exposure resulted in significant impairment of fracture callus tissue formation, perturbation of the key Wnt pathway protein ß-catenin, and disruption of normal Wnt-mediated transcription. These data suggest that the canonical Wnt pathway is a target for alcohol in bone and may partially explain why impaired fracture healing is observed in alcohol-abusing individuals.

Antioxidant therapy attenuates deficient bone fracture repair associated with binge alcohol exposure.

OBJECTIVES: Alcohol consumption is a known risk factor for traumatic injuries of all types and has been shown to produce detrimental effects on bone metabolism. Although the mechanisms responsible for these detrimental effects are not well characterized, oxidative stress from alcohol exposure appears to play a central role. This study was designed to examine the effect of a short-term binge alcohol consumption pattern on fracture repair and the effect of an antioxidant, N-acetylcysteine, on fracture healing after binge alcohol consumption. METHODS: One hundred forty-four adult male Sprague-Dawley rats underwent unilateral closed femur fracture after injection of either saline or alcohol to simulate a binge alcohol cycle. Animals in the antioxidant treatment group received daily N-acetylcysteine after fracture. Femurs were harvested at 1, 2, 4, and 6 weeks after injury and underwent biomechanical testing and histologic analysis. RESULTS: Binge alcohol administration was associated with significant decreases in biomechanical strength at 1- and 2-week time points with a trend toward decreased strength at 4- and 6-week time points as well. Alcohol-treated animals had less cartilage component within the fracture callus and healed primarily by intramembranous ossification. Administration of N-acetylcysteine in alcohol-treated animals improved biomechanical strength to levels comparable to the control animals and was associated with increased endochondral ossification. CONCLUSIONS: Our results indicate that binge alcohol alters the quality of fracture healing after a traumatic injury and that concurrent administration of an antioxidant is able to reverse these effects.

Binge alcohol exposure modulates rodent expression of biomarkers of the immunoinflammatory response to orthopaedic trauma.

BACKGROUND: Alcohol is a known modulator of the immune system and host-defense response. Alcohol abuse is common in trauma patients, although the influence of alcohol intoxication on the inflammatory response following major orthopaedic injury remains unknown. The aim of this investigation was to examine the influence of binge alcohol exposure on biomarkers of the systemic inflammatory response following bilateral traumatic femoral fracture in a rodent model. METHODS: Ninety-two Sprague-Dawley rats were administered intraperitoneal injections of either saline solution or alcohol for three days. These animals then underwent a sham procedure or bilateral femoral intramedullary pinning and mid-diaphyseal closed fracture via blunt guillotine. The animals were killed at specific time points after the injury. Serum and lung tissue were collected, and twenty-five inflammatory markers were analyzed by immunoassay. Histological sections of lung tissue were evaluated by a board-certified pathologist. RESULTS: Bilateral femoral fracture significantly (p < 0.05) increased multiple serum biomarkers of inflammation. Binge alcohol treatment prior to injury significantly suppressed the increase in serum levels of interleukin (IL)-6, white blood cells, IL-2, IL-10, and C-reactive protein after the fracture. However, alcohol-treated animals were found to have increased pulmonary levels of IL-6, IL-1ß, IL-2, and macrophage inflammatory protein-1α following bilateral femoral fracture. In addition, lung tissue harvested following alcohol treatment and injury demonstrated increased pathologic changes, including parenchymal, alveolar, and peribronchial leukocyte infiltration and significantly elevated pulmonary wet-to-dry ratio, indicative of pulmonary edema. CONCLUSIONS: Our results indicate that acute alcohol intake prior to bilateral femoral fracture with fixation in rats modulates the inflammatory response after injury in a tissue-dependent manner. Although serum biomarkers of inflammation were suppressed in alcohol-treated animals following injury, several measures of pulmonary inflammation including cytokine levels, histological changes, and findings of pulmonary edema were significantly increased following fracture with the presence of alcohol.

Alcohol exposure and mechanisms of tissue injury and repair.

Tissue injury owing to acute and chronic alcohol consumption has extensive medical consequences, with the level and duration of alcohol exposure affecting both the magnitude of injury and the time frame to recovery. While the understanding of many of the molecular processes disrupted by alcohol has advanced, mechanisms of alcohol-induced tissue injury remain a subject of intensive research. Alcohol has multiple targets, as it affects diverse cellular and molecular processes. Some mechanisms of tissue damage as a result of alcohol may be common to many tissue types, while others are likely to be tissue specific. Here, we present a discussion of the alcohol-induced molecular and cellular disruptions associated with injury or recovery from injury in bone, muscle, skin, and gastric mucosa. In every case, the goal of characterizing the sites of alcohol action is to devise potential measures for protection, prevention, or therapeutic intervention.

Long-term modulations in the vertebral transcriptome of adolescent-stage rats exposed to binge alcohol.

AIMS: Dangerous alcohol consumption practices are common in adolescents, yet little is known about their consequences on attainment of peak bone mass and long-term skeletal integrity. We previously demonstrated that binge alcohol-exposed adolescent rats showed site-specific reductions in accruement of bone mineral density and bone strength, which were incompletely recovered following prolonged alcohol abstinence. Currently, we analysed the vertebral transcriptome of adolescent rats following alcohol treatment and abstinence to identify long-term molecular changes in the lumbar spine. METHODS: Sixty male adolescent Sprague-Dawley rats were assigned to one of six treatment groups receiving binge alcohol (3 g/kg) or saline i.p., 3 consecutive days (acute binge), 4 consecutive weekly (3-day) binge cycles (chronic binge) or 4 weekly binge cycles followed by a 30-day abstinence period (chronic binge with abstinence). Following treatment, lumbar vertebrae were assayed for global transcriptional changes using gene array technology. RESULTS: Analysis of the adolescent rat vertebral transcriptome identified clusters of binge alcohol-sensitive genes displaying differential expression patterns starting before bone damage was seen and persisting after alcohol treatment was discontinued. Functional grouping of these gene clusters identified candidate cellular pathways affected following acute and chronic binge treatment, as well as pathways remaining modulated following abstinence. CONCLUSIONS: These results demonstrate that binge alcohol exposure can produce disruptions of normal bone gene expression patterns in the adolescent rat that persist well beyond the period of active intoxication. This data may have relevance to peak bone mass attainment and future risk of skeletal disease in adolescents engaging in repeated binge-drinking episodes.

Correlation of measurable serum markers of inflammation with lung levels following bilateral femur fracture in a rat model.

INTRODUCTION: Evaluation of the systemic inflammatory status following major orthopedic trauma has become an important adjunct in basing post-injury clinical decisions. In the present study, we examined the correlation of serum and lung inflammatory marker levels following bilateral femur fracture. MATERIALS AND METHODS: 45 Sprague Dawley rats underwent sham operation or bilateral femoral intramedullary pinning and mid-diaphyseal closed fracture via blunt guillotine. Animals were euthanized at specific time points after injury. Serum and lung tissue were collected, and 24 inflammatory markers were analyzed by immunoassay. Lung histology was evaluated by a blinded pathologist. RESULTS: Bilateral femur fracture significantly increased serum markers of inflammation including interleukin (IL)-2, IL-6, IL-10, GM-CSF, KC/GRO, MCP-1, and WBC. Femur fracture significantly increased serum and lung levels of IL-1a and KC/GRO at 6 hours. Lung levels of IL-6 demonstrated a trend towards significance. Histologic changes in pulmonary tissue after fracture included pulmonary edema and bone elements including cellular hematopoietic cells, bone fragments and marrow emboli. DISCUSSION AND CONCLUSION: Our results indicate that bilateral femur fracture with fixation in rats results in increases in serum markers of inflammation. Among the inflammatory markers measured, rise in the serum KC/GRO (CINC-1), a homolog to human IL-8, correlated with elevated levels of lung KC/GRO. Ultimately, analysis of serum levels of KC/GRO (CINC-1), or human IL-8, may be a useful adjunct to guide clinical decisions regarding surgical timing.

Effect of corticosteroids on the biomechanical strength of rat rotator cuff tendon.

BACKGROUND: The effect of corticosteroids on tendon properties is poorly understood, and current data are contradictory and diverse. The biomechanical effect of steroids on rotator cuff tendon has not been studied, to our knowledge. The current study was undertaken to characterize the biomechanical effects of corticosteroid exposure on both uninjured and injured rat rotator cuff tendon. METHODS: One hundred and twenty-three male Sprague-Dawley rats were randomly assigned to four groups: control (C), tendon injury (I), steroid exposure (S), and tendon injury plus steroid exposure (I+S). Unilateral tendon injuries consisting of a full-thickness defect across 50% of the total width of the infraspinatus tendon were created. Steroid treatment consisted of a single dose of methylprednisolone placed into the subacromial space. At one, three, and five weeks postoperatively, the shoulders were harvested and the infraspinatus tendon was subjected to biomechanical testing. Two specimens from each group were used for histological analysis. RESULTS: At one week, maximum load, maximum stress, and stiffness were all significantly decreased in Group S compared with the values in Group C. Mean maximum load decreased from 37.9 N in Group C to 27.5 N in Group S (p < 0.0005). Mean maximum stress decreased from 18.1 MPa in Group C to 13.6 MPa in Group S (p < 0.0005). Mean stiffness decreased from 26.3 N/mm in Group C to 17.8 N/mm in Group S (p < 0.0005). At one week, mean maximum stress in Group I+S (17.0 MPa) was significantly decreased compared with the value in Group I (19.5 MPa) (p < 0.0005). At both the three-week and the five-week time point, there were no significant differences between Group C and Group S or between Group I and Group I+S with regard to mean maximum load, maximum stress, or stiffness. Histological analysis showed fat cells and collagen attenuation in Groups S and I+S. These changes appeared to be transient. CONCLUSIONS: A single dose of corticosteroids significantly weakens both intact and injured rat rotator cuff tendons at one week. This effect is transient as the biomechanical properties of the steroid-exposed groups returned to control levels by three weeks.