ToxSci Advance Access originally published online on October 6, 2004
Toxicological Sciences 2004 82(2):656-660; doi:10.1093/toxsci/kfi002
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Toxicological Sciences vol. 82 no. 2 © Society of Toxicology 2004; all rights reserved.
IL-1 and TNF Antagonists Prevent Inhibition of Fracture Healing by Ethanol in Rats
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* Laboratory for Limb Regeneration Research, Arkansas Children's Hospital Research Institute, Little Rock, Arkansas 722021; Department of Physiology and Biophysics, The Center for Orthopaedic Research, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; Department of Inflammation Research, Amgen, Inc., Thousand Oaks, California 91320; ¶ Department of Pediatrics, || Department of Orthopaedic Surgery, ||| Department of Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and |||| Arkansas Children's Nutrition Center, Little Rock, Arkansas 72202
Received June 1, 2004; accepted September 22, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We tested the hypothesis that combined administration of IL-1 and TNF antagonists would protect fracture healing from inhibition by chronic ethanol exposure. Adult male rats were fed a liquid diet ± ethanol (CON and ETOH) by intragastric infusion for three weeks prior to and three weeks after creation of an externally fixated tibial fracture. Beginning the day of fracture, one-half of each dietary group received 2.0 mg/kg/day IL-1ra and 2.0 mg/kg/2-days sTNFR1 (CON + ANTAG and ETOH + ANTAG), while all other animals received vehicle alone (CON + VEH and ETOH + VEH). Scoring of ex vivo radiographs and analysis by pQCT revealed a significantly lower incidence of bridging and reduced total mineral content in the ETOH + VEH group compared to all other groups. These results support, for the first time, the hypothesis that IL-1 and TNF antagonists are capable of protecting fracture healing from the inhibition associated with chronic ethanol consumption.
Key Words: alcohol; tumor necrosis factor; interleukin-1; interleukin-1 recombinant antagonist; repair; total enteral nutrition.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chronic alcohol consumption is known to disrupt skeletal homeostasis and repair in multiple ways (Bikle, 1993
; Brown et al., 2002; Chakkalakal et al., 2002; Purohit, 1997; Turner, 2000). Clinically, chronic alcoholics show a marked impairment in bone formation (Crilly et al., 1988) and a significant increase in their risk of non-union after fracture (Nyquist et al., 1997). Similar phenomena have been reported in rodents, including ethanol-related decreases in bone formation rate (Turner et al., 1987), osteoblast proliferation (Dyer et al., 1998), and delays in fracture healing (Chakkalakal et al., 2002; Elmali et al., 2002; Nyquist et al., 1999). In addition, at least one group has demonstrated ethanol-induced osteoclastogenesis in mice that appears to be dependent on induction of IL-6 (Dai et al., 2000).
Using the Total Enteral Nutrition (TEN) system of intragastric dietary delivery, this laboratory demonstrated that combined administration of IL-1 and TNF antagonists protected bone formation during distraction osteogenesis (DO) from ethanol-related inhibition (Perrien et al., 2002). It was later reported that the immunohistochemical expression of TNF-
but not IL-1ß was significantly increased in the DO gap, and the mRNA levels of both cytokines were increased in the marrow of ethanol-fed rats (Perrien et al., 2003). Together, these studies provided the first evidence that one or both of these cytokines may play a role in the etiology of ethanol-related skeletal pathology.
Distraction osteogenesis is a variant of fracture healing in which an externally fixated fracture is literally stretched along the long axis of the bone in order to achieve limb lengthening. Obviously, there is a great deal of similarity between the cellular and molecular processes in both of these settings. This is especially true during the early inflammatory phases. However, once the lengthening (distraction) begins during DO, dramatic differences in tissue composition and behavior begin to take place. Most notably, the early cartilaginous/endochondral component of the fracture callus especially in the endosteal compartment is replaced by proliferative fibroblasts and osteoblasts as the mode of ossification becomes dominated by the intramembranous component.
The current study was designed to determine if the previous findings using the DO model are also applicable to the more commonly used rat model of fixated fracture. While these two models are similar, they do harbor several key differences that may have significant effects on the roles of numerous signaling pathways. Unfortunately, the differences in sensitivity to specific molecular signals during DO or fracture repair are poorly understood.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fifty virus-free adult male Sprague Dawley rats (three-months old, 350 g) were purchased from Harlan Industries (Indianapolis, IN). They were housed in individual cages in temperature (22°C) and humidity (50%) controlled rooms having a 12 h light/12 h dark cycle. The rats were assigned to respective experimental groups with mean body weights equal to that of the control group (±4 g) and weighed twice a week thereafter. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences and Arkansas Children's Hospital Research Institute.
All surgical procedures for TEN have been described in detail elsewhere (Badger et al., 1993; Brown et al., 2002). All rats were handled by animal care personnel for 5–7 days prior to surgery. Briefly, a small silicone cannula was inserted through the wall of the stomach and tunneled subcutaneously to the head. It was then attached to a headpiece that was tethered to the top of the cage for infusion of the liquid diet. During the same surgery, stainless steel Illizarov-style ring fixators were applied to the left tibiae in standardized fashion (Aronson et al., 2001; Brown et al., 2002). All rats received 0.1 mg/kg buprenex for analgesia and were returned to their cages for observation during recovery from anesthesia. Dietary infusion began no sooner than 4 h after recovery from anesthesia. Sterile techniques were used to prepare all solutions, and diet was infused at a rate of 187 kcal/kg0.75/day. Water was available ad libitium throughout the study. All rats were acclimated to the TEN system over a three-day period by gradually increasing the rate of dietary infusion to 187 kcal/kg0.75/day. Rats assigned to the EtOH groups were then acclimated to increasing amounts of ethanol (9–12 g/kg/day) in their diet over a one-week period and then maintained at 12 g/kg/day for the remaining five weeks of the study. For each calorie of ethanol added to the diet an isocaloric amount of carbohydrates was removed. Urine ethanol concentrations (UECs), which correlate positively with blood alcohol content, were measured daily for the duration of the study as previously described (Badger et al., 1993). All rats assigned to the control groups were maintained on the control (non-alcoholic) diet for the duration of the experiment.
Three weeks after placement of the intragastric cannula, all rats underwent a second surgery for creation of a transverse mid-diaphyseal osteotomy and implantation of an Alzet mini-osmotic pump (Durect, Cupertino, CA) for drug delivery as described in detail elsewhere (Perrien et al., 2002). At that time the CON and ETOH rats were further subdivided into two additional groups according to drug treatment: CON + VEH, CON + ANTAG, ETOH + VEH, ETOH + ANTAG. Under isofluorane anesthesia, three holes were drilled in the mid-diaphysis of the fixated left tibia. Direct lateral pressure was then applied to the weakened site to create the standardized fracture. For pump implantation, a 1 cm lateral subcutaneous incision was made on the back just above the scapulae. A small pocket was created using a blunt instrument, and an Alzet model 2ML2 osmotic pump delivering either 1.8 mg/kg/day IL-1ra (Amgen, Thousand Oaks, CA) (CON + ANTAG and ETOH + ANTAG) or vehicle (CON + VEH and ETOH + VEH) alone was inserted. After closing the incision, each animal received an im injection of 0.1 mg/kg buprenex for analgesia. In addition, rats in the antagonist treated groups received 2.0 mg/kg PEG-r-met-sTNFR1 (sTNFR1; Amgen) via sc injection at the time of surgery and every other day thereafter. Vehicle treated rats received equivalent sc injections of saline. Injections were given on alternating sides to minimize stress to the animals. After a 21-day healing period, the rats were euthanized under anesthesia. The operated tibiae were surgically removed by disarticulation at the knee and ankle and cleaned of all soft tissue prior to radiographic imaging and storage in 10% neutral buffered formalin.
Peripheral quantitative computerized tomography (pQCT) analysis. The excised tibiae were scanned by pQCT (XCT research SA, Norland Medical Systems, Fort Atkins, WI) using the manufacturer's software version 5.40 for slice reconstruction and analysis. A single cross-section of the central fracture callus between the proximal and distal host cortices was obtained with a voxel size of 110 µm. A threshold of 214 mg/cm3 was used to distinguish new bone from soft tissue within the callus. The total volumetric mineral content, density, and area of new bone were determined (Ke et al., 2001). Using these threshold settings, it was determined that the ex vivo precision of volumetric mineral content, density, and area of new bone ranged from 0.99 to 3.48% with repositioning.
Micro-computed tomography (µCT). Representative fracture calluses were selected based on standard radiographs and scanned in a µCT40 microCT unit (Scanco Medical, Bassersdorf, Switzerland). In order to visualize the entire callus at sufficiently high resolution, each specimen required acquisition of 700–900 cross sectional slices with a voxel size of 17 µm in all directions. The manufacturer's software was then used to define a volume of interest on the two-dimensional slices. The slices were subsequently stacked into a three-dimensional cube before reapplying the VOI boundaries, an optimized grey scale threshold, and a Gaussian noise filter to create a binary three-dimensional rendering of the bony tissue.
Statistical methods. Incidence of radiographic union was compared by z-test of proportions. pQCT data was analyzed by one-way ANOVA and Tukey post hoc tests. Results were considered statistically significant when p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All groups gained weight at an equivalent and steady rate throughout the study period. The weights at sacrifice between the control and ethanol fed rats were not significantly different (control = 385 ± 6.7 g, ethanol = 370 ± 7.6 g). In the rats fed a diet containing ethanol, the UECs varied with the established pulsatile pattern from 100 to 550 mg/dl (average 206 mg/dl). Of the 56 rats that began the study 18 were sacrificed prior to completion of the protocol due to failure of the intragastric cannula, surgical complications, or other health problems. Consequently, these animals were excluded from all analyses reducing the final size of the groups to n = 11 for CON + VEH, n = 10 for CON + ANTAG, n = 8 for ETOH + VEH, and n = 9 for ETOH + ANTAG.
As a clinically relevant measure of repair, standard two-dimensional radiographs of the excised fracture calluses were qualitatively scored for presence or absence of radiographic union by a blinded, trained observer. Analysis revealed that, after 21 days of healing, fracture calluses from ETOH + VEH rats had a significantly lower incidence of radiographic union compared to all three other groups (p < 0.05; Table 1). The incidence of radiographic union was not significantly different between the remaining three groups.
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Quantitatively, pQCT was used to measure the volumetric, mineral content, density, and volume of new bone in the central fracture callus, the same region scored for bridging. This analysis produced similar results to those found in the qualitative bridging analysis. Similar to the results of the bridging analysis, quantification of the mineral content showed the ETOH + VEH rats had significantly less mineral in the central callus than either CON + VEH or CON + ANTAG rats (p < 0.05 for both), which were not significantly different from each other. In contrast, the mineral content of ETOH + ANTAG rats was not significantly different from any of the other three groups. While the differences in mineral content were the only measure to reach statistical significance (p < 0.05; Fig. 1), a similar trend was seen for both density and new bone volume. Again, this demonstrates that, as expected, ethanol exposure impairs fracture healing in vehicle treated rats, and treatment of ethanol fed but not control fed rats with IL-1 and TNF antagonists can enhance the process (Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Together, these results indicate that inclusion of ethanol in the diet of vehicle treated rats impaired fracture repair by reducing the likelihood of radiographic union and the mean volumetric mineral content at 21 days. More importantly, the significantly higher incidence of union in ETOH + ANTAG rats compared to ETOH + VEH treated rats and the lack of a significant difference in mineral content between the ETOH + ANTAG and CON + VEH groups suggests that antagonism of IL-1 and TNF signaling was able to protect the repair process from the inhibitory effects of ethanol exposure. In addition, the apparent similarity between the CON + VEH and CON + ANTAG groups also indicates that the impact of the antagonists on fracture healing is specific to the condition of ethanol exposure. This last conclusion is of particular importance as it implies that IL-1 and/or TNF signaling may be of particular importance in ethanol-related skeletal pathology and this is supported by previously published data by this (Perrien et al., 2002
, 2003) and other laboratories (Turner et al., 1998). Specifically, rats fed an ethanol containing diet using either the TEN system or the Lieber-DeCarli paired feeding system, have increased expression of TNF- and/or IL-1ß in femoral marrow samples (Perrien et al., 2003; Turner et al., 1998). In addition, this laboratory has documented increased expression of TNF- in osteoblasts in response to ethanol exposure in vitro and in vivo in ethanol fed rats undergoing distraction osteogenesis (Perrien et al., 2003). Thus, it is possible that, in the early phase of fracture healing when the newly forming fracture gap is contiguous with the bone marrow, excess ethanol induced TNF in the marrow cells (stromal cells, T cells, etc.) negatively affects bone formation. Once the fracture architecture is formed, then direct ethanol induction in the fracture cells (stromal cells, pre-osteoblasts, osteoblasts, chondroblasts, etc.) of excess TNF is a possible source of osteoinhibition. Also, since mature fractures are well vascularized a contribution of mononuclear cells is still possible. Recent papers suggest another level of complexity, that peripheral blood mononuclear cells were shown to secrete TNF after co-culture with a stromal-like cell line (Atkins et al., 2000; Nanes, 2003). Thus, cytokine over production can also be modulated by cell-cell contact and this possibility should be considered in this context. In conclusion, the results of the current study are in line with previously published data and add to a growing body of evidence that induction of TNF- and/or IL-1ß is at least partially responsible for ethanol-induced skeletal pathology. |
ACKNOWLEDGMENTS |
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Supported in part by NIH grants AA12223 and AA08645, and NCRR grant RR16517.
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NOTES |
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1 To whom correspondence should be addressed at ACHRI, Slot 512-20B, 1120 Marshall Street, Little Rock, AR 72202. Fax (501) 364-5880. E-mail: lumpkincharlesk{at}uams.edu.
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