* 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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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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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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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 57 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 (912 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 700900 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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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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DISCUSSION |
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ACKNOWLEDGMENTS |
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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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