ArticleInhibition of bone repair in a rat model for chronic and excessive alcohol consumption
Introduction
Higher incidences of complications were encountered during fracture treatment in patients who are classified as alcoholics (Adell et al., 1987, Nyquist et al., 1997, Passeri et al., 1993, Tonnesen et al., 1991). It is often assumed that these complications result mostly from noncompliance of the patients with the protocols. However, some studies using ethanol-fed rat models suggest that alcohol may have a more direct biological effect on the process of fracture healing (Brown et al., 2002, Chakkalakal et al., 2002, Elmali et al., 2002, Janicke-Lorenz and Lorenz, 1984). Ethanol has indeed been shown to have an antiproliferative effect on osteoblastic cells in culture (Chavassieux et al., 1993, Friday and Howard, 1991) and in ethanol-fed rats (Dyer et al., 1998). It also inhibits osteoblast function in vitro (Chavassieux et al., 1993, Farley et al., 1985, Friday and Howard, 1991, Hurley et al., 1990, Klein et al., 1996) and in vivo in rats (Dyer et al., 1998) and human beings (Diez et al., 1997, Garcia-Sanchez et al., 1995).
Earlier experimental studies have provided mixed results on the effect of ethanol on fracture healing. In a qualitative study, rats that underwent osteotomy of both femora after 1 year of ethanol consumption had a total absence of fracture callus as revealed by radiographs at 9 weeks postosteotomy when femora in ethanol-free controls had completely healed with mineralized callus (Janicke-Lorenz & Lorenz, 1984). In contrast, Pierce and Perry (1991) found that rats consuming ethanol during 5 weeks of fracture healing had significantly higher breaking strength of fractured tibia than ethanol-free controls at 5 weeks. On the other hand, Nyquist et al. (1999) found that rats given ethanol during 1 week before and 5 weeks after fracture of tibia demonstrated inferior biomechanical properties and mineralization than controls receiving no ethanol at 5 weeks postfracture. But they also found similar reductions in these properties for the nonfractured contralateral tibiae and hence concluded that ethanol did not have a specific effect on the fracture healing process.
More recent studies have provided additional evidence for adverse effects of ethanol on fracture healing. In a previous study in ethanol-fed rats (Chakkalakal et al., 2002), we found that 13 weeks of alcohol feeding (6 weeks before and 7 weeks after bone injury) produced bone repair tissue of lower stiffness, but unchanged ash density, in a gap osteotomy bridged with a demineralized-bone-matrix (DBM) scaffold, when compared with pair-fed controls receiving no ethanol. Elmali et al. (2002) found that repair tissue in tibial fractures fixed with intramedullary nail in rats given ethanol for 8 weeks (4 weeks before and 4 weeks after fracture) was poorly mineralized and fibrous, whereas in pair-fed controls the repair tissue consisted of lamellar bone and calcified cartilage undergoing ossification indicating normal healing. Lastly, Brown et al. (2002) reported that ethanol inhibited the rapid “intramembranous” bone formation, which characterizes normal healing of the osteotomy gap in a “distraction osteogenesis” model in rats.
It is difficult to determine definitely whether ethanol inhibits fracture healing by taking these studies together, mainly because of methodological differences, most notably, in fracture model, ethanol dose, method of feeding, and duration of feeding before and after fracture. Therefore, we designed the present study to answer two questions: (1) Is there a dose-dependent deficiency in bone repair outcome associated with ethanol consumption? (2) Does the reduced food intake often associated with chronic consumption of excessive alcohol by human beings, also influence this outcome? We used ethanol as 36% of total calories in the diet as a “high” dose because, in rats it is known to produce high-blood ethanol levels (26–43 mM), behavioral intoxication, and certain characteristics of abnormal liver metabolism that precede the onset of human alcoholic liver disease (Donohue et al., 1987, Hunter et al., 1975). This dose has also been used in several studies of ethanol-induced osteopenia in rat skeleton (Turner, 2000). We used ethanol as 26% of total calories as a “moderate” dose because in the rat model this dose results in very low levels of blood ethanol and no fatty liver (Rao et al., 1987). The purpose of using these two doses is to determine whether the ethanol-induced deficiency in bone repair outcome is dose dependent. Alternately, we will determine whether the “moderate” dose has a beneficial effect on bone repair, analogous to the reported improvement of bone mineral density in postmenopausal women (Felson et al., 1995). The bone repair model in our previous study (Chakkalakal et al., 2002), described briefly in Section 2, is used in the present study, but with a more thorough determination of the mechanical properties of the “fractured” bone as a whole and the repair tissue. Another limitation of the previous study was that we could not determine whether reduced intake of the ethanol-free (control) liquid diet or the difference between this diet and standard rat chow contributed to deficient bone repair. The present study design includes the necessary controls to allow this determination.
Section snippets
Research design
We used two ethanol-feeding models in which 11- to 12-week-old male Wistar rats were given Lieber–DeCarli liquid diets (Lieber & DeCarli, 1982) containing ethanol as 36% and 26% of total calories denoted by LDE 36 and LDE 26, respectively. Ethanol-free control diets corresponding to LDE 36 and LDE 26 are denoted by LDC 36 and LDC 26, respectively. The corresponding models are designated as “high-ethanol consumption” (HEC) model and “moderate ethanol consumption” (MEC) model. The HEC model
Results
The animals were healthy throughout the protocol, and there were no infections at the wound site or elsewhere. There was no noticeable difference in the physical activity of the ethanol-fed and control rats or between rats in the HEC and MEC models. This was true both before and after surgery. In group G, in which rats were given ethanol as 26% calories for 13 weeks, there was loss of one animal due to premature death. This animal was excluded from data analysis.
Discussion
The main findings in this study are as follows: (1) consumption of “excessive” ethanol (as 36% of total calories) during 6 weeks before and 7 weeks after bone injury (HEC model) was associated with a deficient bone repair outcome, whereas “moderate” consumption (i.e., 26% of total calories) had little or no effect; (2) the deficiency was caused by ethanol and not the reduced food intake by ethanol-fed rats; and (3) in the HEC model, withdrawal of diet containing ethanol after bone injury and
Acknowledgments
We thank Veronique Pileri for assistance in the rat feeding protocol. This work was supported by a Merit Review grant from the U.S. Department of Veterans Affairs. Partial support was provided by a Health Future Foundation grant to Creighton University Biomedical Engineering Research Center.
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