The Effect of Ethanol on Rotator Cuff Repairs in a Rodent Model.

BACKGROUND: Alcohol consumption is a significant risk factor for both the occurrence and severity of rotator cuff tears. However, there is limited supporting evidence to suggest alcohol use is associated with suboptimal outcomes following operative repair of rotator cuff tears. Rat shoulders have been demonstrated as consistent and reliable models for studying rotator cuff disease. HYPOTHESIS/PURPOSE: Perioperative alcohol exposure will negatively impact biomechanical and histologic properties of surgically repaired rotator cuffs in rats. METHODS: Rats were randomized to receive a 20% ethanol or isocaloric control solution as their primary source of drinking water. A tenotomy of the supraspinatus tendon from bone was performed surgically and then immediately repaired with a transosseous technique. Following surgery, rats were continued on the same exposure solution until animals were humanely euthanized at 7, 14, or 21 days postoperatively. The surgically-repaired shoulders underwent biomechanical testing to assess load to failure and failure strain. Histological evaluation of tendon-to-bone healing was performed by a blinded pathologist using a qualitative grading system. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on total RNA from tendon-to-bone interface tissue was performed to quantify the mRNA expression of Type I & III collagen, and transforming growth factor-beta 1 (TGF-B1) & 3 (TGF-B3) at the repair site. RESULTS: Biomechanical testing showed that repaired shoulder constructs in rats exposed to ethanol had significantly lower load to failure at 7 days postop relative to repairs in rats exposed to a control solution. No other biomechanical parameters or time points reached statistical significance. TGF-B3 mRNA expression was found in significantly higher quantities at the repair sites of rats exposed to ethanol at 7 days postop relative to control rat repair sites. No other time points or factors reached statistical significance. No significant differences were identified amongst time points or groups at the healing tendon-to-bone interface. CONCLUSION: Alcohol exposure significantly decreases biomechanical load to failure of rotator cuff repairs in the early postoperative period in rat models. In the later postoperative period, alcohol exposure was not associated with a decrease in biomechanical load to failure compared to controls. Additionally, rats exposed to ethanol have significantly higher TGF-B3 expression at repair sites on postoperative day 7. This data suggests that ethanol consumption does deleteriously affect rotator cuff and bone healing. Future study is needed to validate the clinical significance of these findings in humans.

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.

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.

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.

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.

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.

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.

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.

Binge alcohol-induced bone damage is accompanied by differential expression of bone remodeling-related genes in rat vertebral bone.

Binge alcohol-related bone damage is prevented by concurrent administration of bisphosphonates, suggesting an activation of bone resorption with patterned alcohol exposure. Although chronic alcohol abuse is known to cause osteopenia, little is known about the effects of binge drinking on bone metabolism. We examined the effects of binge alcohol exposure on the relationship between bone damage and modulation of bone remodeling-specific gene expression profiles. Our hypothesis was that bone damage observed in young adult rats after binge alcohol exposure is associated with differential expression of bone remodeling-related gene expression. We further hypothesized that this differential gene expression specific to bone remodeling (bone resorption or formation related) would be influenced by the duration of binge alcohol exposure. Binge alcohol (3 g/kg, i.p.) was administered on 3 consecutive days each week, for 1 or 4 weeks, to adult male rats. Matched control animals were injected with an equal volume of isotonic saline. Lumbar vertebrae, L4-5, were analyzed for the presence of bone damage by quantitative computed tomography and compressive strength analysis. Total RNA was isolated from an adjacent vertebrae (L3), and whole transcriptome gene expression data were obtained for each sample. The expression levels of a subset of bone formation and resorption-associated differentially expressed genes were validated by quantitative reverse transcriptase-polymerase chain reaction. Bone loss was not observed after 1 week of treatment but was observed after four binge alcohol cycles with a 23% decrease in cancellous bone mineral density and 17% decrease in vertebral compressive strength compared with control values (P < 0.05). We observed that the duration of binge alcohol treatment influenced the modulation of expression profiles for genes that regulate the bone formation process. The expression of key bone formation-related marker genes such as osteocalcin and alkaline phosphatase were significantly reduced (P < 0.05) after acute binge alcohol exposure, and expression of regulators of osteoblast activity such as bone morphogenetic proteins and parathyroid hormone receptor displayed significantly (P < 0.05) decreased differential expression. The expression of sclerostin, a key canonical Wnt inhibitory protein, was significantly increased after acute binge alcohol treatment. The expression of important regulators of osteoclast maturation and activity such as NF-kappabeta (nuclear factor kappabeta) ligand (RANKL) and interleukin-6 were significantly increased (P < 0.05) by binge alcohol, and osteoprotegerin levels were significantly decreased (P < 0.05) in vertebral bone. These results show that expression patterns of several key bone remodeling genes are significantly perturbed by binge alcohol treatment, suggesting that perturbation of gene expression associated with bone remodeling may be one mechanism contributing to the disruption of bone mass homeostasis and subsequent bone loss observed after binge alcohol exposure in rodents.

The effect of corticosteroid on collagen expression in injured rotator cuff tendon.

BACKGROUND: Subacromial corticosteroid injections are commonly used in the nonoperative management of rotator cuff disease. The effects of corticosteroid injection on injured rotator cuff tendons have not been studied. Our aims were to characterize the acute response of rotator cuff tendons to injury through the analysis of the type-III to type-I collagen expression ratio, a tendon injury marker, and to examine the effects of corticosteroid on this response. METHODS: Sixty Sprague-Dawley rats were randomly assigned to four groups: control, tendon injury, steroid treatment, and tendon injury and steroid treatment. Six rats served as sham controls. Unilateral tendon injuries were created with full-thickness defects across 50% of the total width of the infraspinatus tendon, 5 mm from its humeral insertion. Steroid treatment with a single dose of methylprednisolone (0.6 mg/kg), equivalent to that given to humans, was injected into the subacromial space under direct visualization. Steroid treatment followed the creation of an injury in the rats in the injury and steroid treatment group. At one, three, and five weeks after the injury, the total RNA isolated from tendons was quantified with real-time polymerase chain reaction with use of primers for type-I and type-III collagen and ribosomal 18s RNA. RESULTS: The type-III to type-I collagen expression ratio remained at baseline at all time-points in the control and sham groups. At one week, the type-III to type-I collagen expression ratio increased more than fourfold above the control level in the tendon injury group (p = 0.017) and the tendon injury and steroid treatment group (p = 0.003). The ratio remained greater than twofold above the control at three weeks in both groups (p = 0.003 and p = 0.037) and returned to baseline at five weeks. Interestingly, the group that had steroid treatment only showed an increase of >4.5-fold (p = 0.001) in the type-III to type-I collagen expression ratio, without structural injury to the tendon. This ratio returned to baseline levels by three weeks. CONCLUSIONS: A single dose of corticosteroid does not alter the acute phase response of an injured rotator cuff tendon in the rat. However, the same steroid dose in uninjured tendons initiates a short-term response equivalent to that of structural injury.