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.

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.

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.

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.

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.

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.

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 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.

Identification of novel bone-specific molecular targets of binge alcohol and ibandronate by transcriptome analysis.

BACKGROUND: Our laboratory established that binge alcohol-related bone damage is prevented by aminobisphosphonates, suggesting bone resorption increases following binge exposure. We examined the effects of binge alcohol and antiresorptive therapy on the relationship between bone damage and modulation of the vertebral transcriptome, in an attempt to determine how alcohol-induced bone damage and its prevention modulate bone-related biological pathways. METHODS: Male Sprague-Dawley rats were assigned to 1 of 6 treatment groups (n = 12/group). (C1) saline ip 3 d/wk for 1 week, (A1) binge alcohol, 3 g/kg, ip 3 d/wk for 1 week, (C4) saline ip, 3 d/wk for 4 weeks, (A4) binge alcohol, ip, 3 g/kg 3 d/wk for 4 weeks, (I4) ibandronate, saline ip 3 d/wk for 4 weeks, plus a single ip injection of ibandronate at 120 microg/animal, and (AI4) binge alcohol plus ibandronate as above. After 1 or 4 weeks, adjacent lumbar vertebrae were assayed for bone damage or transcriptional changes. RESULTS: Bone loss was not observed after 1 week of binge alcohol treatment. After 4 weeks, binge alcohol decreased vertebral BMD by 23% (p < 0.05) and compressive strength by 18% compared to saline controls (p < 0.05). Concurrent ibandronate prevented bone loss, increasing these parameters by 145 and 134% respectively compared to binge alcohol. (p < 0.05). Analysis of the vertebral transcriptome identified gene clusters specific for acute and chronic binge alcohol-related bone damage. Acute binge alcohol modulated the expression of integrin signaling-specific genes, while chronic binge alcohol modulated canonical Wnt signaling gene expression. Ibandronate normalized the expression of approximately 20% of the genes affected by chronic binge alcohol, allowing the identification of a unique subset of alcohol-sensitive, ibandronate-responsive genes. CONCLUSIONS: Identification of bone-specific gene expression clusters associated with acute and chronic binge alcohol treatment allowed for the identification of cellular pathways affected by binge treatment with known involvement in bone remodeling (Integrin, Canonical Wnt signaling) not previously identified as alcohol-sensitive. This data provides a basis for a plausible mechanistic explanation for the known detrimental effects of alcohol on bone formation and resorption.

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 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.

Vitamin D and ibandronate prevent cancellous bone loss associated with binge alcohol treatment in male rats.

Decreased bone mass and bone strength can result from excess alcohol consumption in humans and alcohol treatment in the rat. Although the specific mechanism is unknown, the damaging effects of alcohol abuse modulate the bone remodeling cycle and increase bone turnover. Chronic alcohol consumption models have shown an inhibition of bone formation. We previously reported that binge alcohol treatment increases bone resorption and that alcohol-induced damage can be prevented by treatments with intermittent parathyroid hormone and bisphosphonates. In this study, we hypothesized that an effective dose of vitamin D (cholecalciferol) or a single dose of ibandronate would prevent bone loss caused by binge alcohol treatment in male rats. Forty-eight adult (450 gram) male Sprague-Dawley rats were randomly assigned to 6 treatment groups (n=8): (a) saline i.p., 3 days/week (C); (b) binge alcohol, 3 g/kg i.p., 3 days/week (A); (c) vitamin D, 5,000 IU/kg daily s.c. (D); (d) binge alcohol and vitamin D (AD); (e) ibandronate (120 microg, given as a single i.p. injection (I)); and (f) alcohol and ibandronate (AI) . After 4 weeks of treatment, proximal tibia and L3 and L4 vertebrae were analyzed for bone mineral density (BMD) by quantitative computerized tomography and compressive strength-to-failure using an Instron materials testing machine. Type I collagen cross-linked c-telopeptide, calcium, and 25-OH vitamin D levels were measured in serum collected at the time of sacrifice. Binge alcohol significantly decreased cancellous BMD by 58% in tibia and 23% in lumbar spine (p<0.05). Binge alcohol treatment decreased L3 and L4 compressive strength-to-failure by 21% (p<.05). Treatment with vitamin D at 5,000 IU/kg/day prevented alcohol-induced bone loss, significantly increasing both tibial and vertebral cancellous BMD values (161% increase in tibia and 40% increase in vertebra, respectively, p<0.05) compared to alcohol alone groups. Pre-treatment with the single dose of 120 microg ibandronate prevented alcohol-induced bone loss, increasing cancellous BMD by 186% in tibiae and by 46% in vertebrae compared to the alcohol alone group (p<0.05). In summary, binge alcohol-induced tibial and vertebral bone loss can be prevented using an effective dose of vitamin D or a single dose of ibandronate even during high blood alcohol concentrations that have been shown to impair osteoblast functions and increase bone resorption.

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.

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.

Spine bone mineral density and vertebral body height are altered by alcohol consumption in growing male and female rats.

Alcohol, consumed for extended periods by growing male and female rats, impairs osteogenesis and reduces bone size and mass. The skeletal sites of experimental animals commonly chosen for an evaluation of bone mechanical characteristics and architectural properties, bone matrix gene expression, tissue concentrations of growth factors, and bone mineral density (BMD) have been the tibiae and femora. Far less attention has been focused on the spine and the effects of alcohol on vertebral BMD and vertebral body height. Fifteen male and 15 female Sprague-Dawley rats (aged 30 days) were divided into three groups: an alcohol-fed group, matched to a pair-fed non-alcohol isocaloric-fed control group with animals of the same sex, and an ad libitum-fed control group. Alcohol-fed animals received a Lieber-DeCarli liquid diet containing 36% of caloric intake as alcohol; isocaloric pair-fed rats received the same diet without alcohol. After 45 days of feeding, the lumbar spine was removed. The fourth lumbar vertebra from each spine was dissected, and the vertebral body height was measured. Lumbar vertebral body height was significantly reduced by alcohol consumption in both male and female rats compared with findings for either control group. Cancellous and cortical BMD of the vertebral body was determined by peripheral quantitative computed tomography (pQCT). Male and female rats (aged 75 days) in the ad libitum-fed group had similar vertebral body cortical and cancellous BMD, with cortical BMD being greater than cancellous BMD. Lumbar vertebral body cancellous and cortical BMD declined for both male and female rats in response to alcohol consumption for 45 days compared with findings for either control group. More BMD loss occurred from cancellous than from cortical bone in both sexes after chronic alcohol consumption. Chronic alcohol consumption by growing rats results in vertebral growth reduction and vertebral osteopenia.

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.