1 Omaha Veterans Affairs Medical Center, Omaha, NE 68105,
2 Creighton University School of Medicine, Omaha, NE 68178 and
3 University of Nebraska Medical Center, Omaha, NE 68198, USA
Received 22 February 2001; in revised form 30 May 2001; accepted 2 July 2001
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ABSTRACT |
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INTRODUCTION |
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The mechanism of alcohol-associated osteopenia appears to involve a direct effect of alcohol on bone cells that leads to suppression of new bone formation, without any indirect or modulating role for mineral regulating hormones (Sampson, 1997). Alcohol has been shown to have an antiproliferative effect on osteoblastic cells in culture (Friday and Howard, 1991
; Chavassieux et al., 1993
) and in ethanol-fed rats (Dyer et al., 1998
). It also inhibits osteoblast function in vitro (Friday and Howard, 1991
; Chavassieux et al., 1993
) and in vivo (Garcia-Sanchez et al., 1995
; Diez et al., 1997
; Dyer et al., 1998
).
We chose to investigate the effect of alcohol on bone healing using rats given ethanol as 36% of total calories as part of a liquid diet, since this experimental model has been used in studies of alcohol-induced bone loss (Baran et al., 1980; Turner et al., 1987
; Peng et al., 1988
; Kusy et al., 1989
; Turner et al., 1991
; Hogan et al., 1997
; Sampson et al., 1997
, 1998
). The liquid diet feeding model demonstrates steatosis and hepatomegaly, which are the early stages of alcoholic liver disease (Donohue et al., 1987
). Bone loss occurs in both the cancellous and compact bone regions in the ethanol-fed rat (Baran et al., 1980
; Peng et al., 1988
; Hogan et al., 1997
; Sampson et al., 1997
, 1998
), as it does in human alcoholics (Bikle et al., 1985
; Diamond et al., 1989
; Peris et al., 1992
). Chronic ethanol feeding of rats causes deficiency in bone matrix synthesis and mineralization, resulting in inferior microstructural architecture and mechanical properties of bone (Baran et al., 1980
; Turner et al., 1987
; Peng et al., 1988
; Kusy et al., 1989
; Turner et al., 1991
; Hogan et al., 1997
; Sampson et al., 1997
, 1998
). These results suggested to us that the repair process in a bone injury in the diaphysis of long bones in these rats may be adversely affected by a similar inhibition of new bone formation which would lead to a deficient bone healing outcome.
The fracture healing process in the long tubular bones of rodents differs from that in humans. In an effort to create a bone repair model in rat long bone that provides a better representation of the human bone healing process than conventional fracture models in the rat, we deliberately altered the bone repair process by surgically creating a segmental defect in rat fibula and fitting it with a tissue scaffold as previously described (Chakkalakal et al., 1999,Chakkalakal et al., 2001
). We have demonstrated the sensitivity of this model to detect the effect of early biological deficiencies on the outcome of bone repair at 7 weeks after injury (Chakkalakal et al., 2000
). The specific objectives of this study were to determine whether: (1) the outcome of bone repair in rats receiving ethanol as part of a nutritionally adequate liquid diet was deficient compared with pair-fed control rats and rats receiving a standard maintenance diet; (2) ethanol affects bone repair in addition to any effect of reduced food intake often associated with ethanol consumption; (3) stopping ethanol consumption after bone injury restores the normal outcome of bone repair or, at least, improves it.
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MATERIALS AND METHODS |
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Surgical procedure
Preoperative and intraoperative anaesthesia of the rats was accomplished by deep intramuscular injection of ketamine (70 mg/kg body weight) and xylazine (3 mg/kg body weight). Details of the surgical procedure that create the bone repair model have been described previously (Chakkalakal et al., 1999, 2001). Briefly, a bilateral osteotomy was performed by exposing the mid-diaphyseal region of the fibulae and removing a 4-mm-long segment including the periosteum. An 8-mm-long tubular specimen of acid-demineralized bone matrix (DBM), prepared in advance from femora of allogeneic animals (Strates and Tiedeman, 1993
), was fitted over the cut ends of the fibula as a tissue scaffold that surrounded the defect. The surgical procedure was performed in all animals 6 weeks after the start of feeding. All animals resumed normal ambulation within 2 days after surgery.
All rats were weighed at the start of the experiment, immediately before surgery and on the day of euthanasia, 7 weeks after surgery. Euthanasia was performed in the morning without a morning feeding. Rats were at first anaesthetized with pentobarbital (50 mg/kg body weight, peritoneally) and a blood sample was obtained from the abdominal aorta for determination of blood-ethanol concentration. Livers were then perfused with ice-cold saline until free of blood. The livers were then removed and either snap-frozen in liquid N2 and stored at 70°C for later use or homogenized immediately for the assay of the ethanol-inducible cytochrome P450 2E1 activity.
Liver and blood alcohol assays
Hepatic cytochrome P450 2E1 (CYP 2E1) activity was measured colorimetrically by the hydroxylation of p-nitrophenol (Koop, 1986). Protein concentrations were determined spectrophotometrically, using the dual wavelength method (Groves et al., 1968
). Weighed pieces of frozen liver (0.360 ± 0.034 g; n = 30) were subjected to total lipid extraction by the method of Folch et al. (1957). Portions of lipid extracts, dissolved in chloroformmethanol (2:1 v/v) were dried under nitrogen at 42°C. The dried extract was saponified at 60°C for 30 min in 90% ethanol containing 0.8 N KOH. Two volumes of 0.15 M MgSO4 were added to the mixtures, which were then centrifuged to precipitate fatty acids. The supernatant fractions were assayed for glycerol (derived from triacylglycerol), using the enzymatic assay kit purchased from Sigma Diagnostics (Cat. #302A) (St Louis, MO, USA). Blood-ethanol concentrations were determined by gas chromatography using the headspace technique (Eriksson et al., 1977
).
Procedures for evaluation of bone repair
The right and left fibulae were excised from all animals immediately after collecting the livers and blood samples. The fibula was separated from the tibia by dissection at the synchondrosis (proximal) and synostosis (distal) with a scalpel and the tibia was discarded. The fibulae were cleaned of adhering soft tissues, and kept moist in normal saline in closed vials. In groups C and D, the outer surface of the DBM scaffold was mineralized and the soft tissue was easily separated without disturbing the bone repair site. In groups A and B, although the outer surface of DBM was less mineralized and hence softer, it was possible to separate the soft tissue from the bone repair tissue, without disturbing the latter, by careful dissection. The fibulae were used for various bone assays as described below.
Mechanical properties of the whole bone and the repair tissue
Three-point bend tests of both fibulae from each rat were performed using methods developed in our laboratory for canine bones (Chakkalakal et al., 1990) and later adapted to rat bones (Chakkalakal et al., 1996
, 1999
). Bend tests were performed on a materials testing machine (Instron Model 1011) within 40 min after the death of the rat, keeping the bones wet in 0.9% saline during this period. The bone was supported on roller supports 15 mm apart with the repair site centred within the test span. Loads of 10200 g were applied, with the load applicator moving at the rate of 1 mm/min. The loaddeflection graph has a linear portion and the test was stopped before reaching the yield point. The vertical diameter (Dv) and the horizontal diameter (Dh) of the fibula at the site of load application were measured using precision calipers. Instron-supplied software was used to calculate rigidity, defined by (Dumbleton and Black, 1981
; Torzilli et al., 1981
):
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Mineral content of the repair tissue
The method used in our laboratory has been previously described (Chakkalakal et al., 1999). After the bend test, a 4-mm segment of the fibula consisting of the entire repair site including the DBM scaffold was excised. These samples were placed in crucibles, dried at 110°C for 24 h, weighed and then ashed at 700°C for 24 h. Ash weight was calculated as a percentage of dry tissue weight.
Statistical analysis
Data analysis was performed using descriptive statistics to determine mean and 95% confidence interval for each variable. To take into account the non-independent status of two samples (fibulae) from the same animal, overall analysis of variance was performed for a one-factor nested design. Multiple pairwise comparisons corresponding to the specific objectives stated in the Introduction were made using the HolmShaffer sequential Bonferroni procedure to maintain experimentwise type I error at the P 0.05 level (Shaffer, 1986
). Comparisons that were not statistically significant are designated n.s.
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RESULTS |
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Gain of body weight during the protocol
The body weights of the four groups of rats at three time points during the protocol (start, surgery and end) and the changes in body weight between these time points are given in Table 1. Rats in pair-fed groups A and B had nearly identical starting body weights due to weight-matching, but this was 12 and 17% lower than in groups C and D respectively. However, at the time of surgery, group D had almost the same average body weight as groups A and B. Only group C, which received the AIN-93M diet, had a larger gain, so that it was significantly higher than the other three groups at the time of surgery. The average rate of weight gain of rats in group D before surgery was 77% less than that of group A rats, in spite of nearly identical rates of consumption of the liquid ethanol diet (55 vs 52 ml/day/rat). During the 7-week post-surgery period, group D rats consumed the control diet at the rate of 89 ml/day/rat compared to 64 ml/day/rat in group A (and B). As a result, the average rate of weight gain of group D rats post-surgery was 2.6-fold that in group A. The overall rate of weight gain during 13 weeks of the protocol was the same for groups A and B, which was significantly lower than that for groups C and D.
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DISCUSSION |
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The most obvious effect of the reduced food consumption by rats in groups A and B is the lower rates of gain of body weight by these animals, compared with those in groups C and D (Table 1, last column). We recognized that the pattern of differences in rigidity of fibula among the four groups (Fig. 1
) closely resembled that of the average body weights of these animals at death (Table 1
, column 4). Therefore, we examined whether the results for rigidity can be explained on the basis of differences in gain of body weight or the amount of food consumed. In a previous study in our laboratory using this bone repair model in chow-fed male SpragueDawley rats, which weighed 330360 g at death, the rigidity was comparable (1.60 ± 0.42 x 103 Nm2, unpublished data) with that of group C (Fig. 1
). Since final body weights of rats in groups A and B in the present study were similar to the previous study, the lower rigidity of fibula in these rats compared with those of group C cannot be attributed to their reduced gain of body weight during the 13-week protocol.
We also examined whether the lower flexural modulus in the ethanol-fed rats in group A compared with pair-fed controls (group B) can be explained in terms of factors other than ethanol. First, there was no difference between these two groups in their average body weights at the start or at the end of the 13-week protocol. Thus, group B proved to be a valid control for the lower body weight gain of ethanol-fed rats in group A associated with their reduced food consumption. In group B, despite the reduced consumption of the Lieber DeCarli control diet imposed by pair feeding and the resultant retardation in body weight gain, the flexural modulus of repair tissue was comparable to that of group C. Although group C received the AIN-93M diet, rather than the LieberDeCarli control diet, the latter is considered to be comparable to the former in nutrition. Indeed, the flexural modulus in group D, which received the LieberDeCarli control diet ad libitum after surgery, is nearly the same as in group C. Therefore neither the reduced food consumption nor the lower body weight at death is a reasonable explanation of the deficiency in flexural modulus of the repair tissue in rats in group A. Thus, we conclude that ethanol was the most likely cause of this deficiency.
The geometric properties of the repaired fibula were also influenced by ethanol consumption. The area of cross-section at the midpoint of the repair site in the fibula of rats in group A was significantly larger than that in group B (Table 3). The area moment of inertia was also larger in group A, but the difference was not statistically significant. Nevertheless, numerically, these results provide a partial explanation for the rigidity of fibula in group B to be the same as group A, in spite of the larger flexural modulus, because of the relationship in equation 4
above. These results suggest a trend in the bone repair process to compensate partially for the inferior mechanical properties of the repair tissue by increasing its bulk as measured by area of cross-section. This, then, would have the effect of mitigating the deficiency in mechanical properties of the bone as a whole and hence in its function.
Comparison of the present study with earlier studies of fracture healing in ethanol-fed rats (Janicke-Lorenz and Lorenz, 1984; Pierce and Perry, 1991
; Nyquist et al., 1999
) may not be valid because of the difference in the method of ethanol feeding. In the earlier studies, rats were fed laboratory chow ad libitum and given 1520% ethanol in the drinking water over periods of 1 year, 8 days and 5 weeks, respectively, before creating the bone injury. If we ignore this difference, the study of Nyquist et al. (1999) comes closest to ours in the duration of feeding before and after bone injury. Although they found significant differences in bending rigidity and strength between ethanol-fed and control rats, they concluded that ethanol had no effect in fracture healing, since they also found similar differences for the non-fractured tibia. However, it must be noted that the presence of a bone injury in the rat is known to influence new bone formation at distant skeletal sites (Mueller et al., 1991
). The study design of Nyquist et al. (1999) does not allow a determination of these effects. Therefore, in view of the statistically significant differences in bending rigidity and strength which they found for fractured tibia, between ethanol-fed and control groups, an effect of ethanol specifically on the fracture healing process in their study cannot be ruled out.
The mineral content of the repair tissue in the pair-fed control rats (group B) was significantly less than in rats receiving the AIN-93M diet ad libitum for 7 weeks after surgery (group D) (Fig. 3). Although there was a further decrease due to ethanol (i.e. from group B to A), it was not statistically significant. In chow-fed rats given ethanol in drinking water, Pierce and Perry (1991) found the mineral content of fractured bones to be nearly the same as in controls not receiving ethanol. These findings suggest that ethanol consumption does not diminish the mineral content of the newly formed tissue in the repair site. However, if ethanol interferes with osteoblast function (Dyer et al., 1998
), it is likely to disrupt the synthesis of an ossifiable matrix resulting in a maldistribution of the mineral. In other words, if dystrophic mineralization (Nimni et al., 1988
), rather than ossification of the newly synthesized matrix, occurs, material properties (e.g. flexural modulus) will be diminished without a decrease in mineral content.
Although the relationship between liver disease and bone disease in alcoholism is not well established, more severe liver disease appears to be associated with greater bone loss (Lalor et al., 1986; Diamond et al., 1989
). In the present study, we observed mild changes in liver, due to alcohol consumption (Table 2
). The 4- to 5-fold elevation in CYP 2E1 activity in livers of ethanol-fed rats confirmed results reported by others, demonstrating that chronic ethanol consumption induces the P450 2E1 isozyme (Buhler et al., 1992
; Akihiko et al., 1995
; Hu et al., 1995
; Wu and Cederbaum, 1996
). The 2.02.4-fold higher levels of hepatic triglycerides in these animals also confirmed previous observations (Donohue et al., 1987
; Guzman and Castro, 1990
). Cessation of ethanol consumption during the 7-week post-surgery period (group D) resulted in normalization of the liver along with the normalization of the bone repair outcome, suggesting a relationship between pathological states in liver and in the bone repair process. Therefore, further studies are warranted using an experimental model in which liver damage is more evident. The intragastric feeding model of Tsukamoto et al. (1985a,b), or the low-carbohydrate oral diet devised by Lindros and Jarvelainen (1998), could be well suited for this purpose, since these models produce more serious liver damage. Either of these models could be used to investigate whether there is a link between liver damage and faulty bone repair.
In conclusion, we found that 13 weeks of feeding ethanol as part of a nutritionally adequate liquid diet (during 6 weeks before and 7 weeks after bone injury) resulted in significant deficiency in the outcome of bone repair compared to ad libitum feeding of a standard maintenance diet. The specific effect of ethanol, apart from any effect associated with reduced food consumption, was to produce repair tissue with inferior material properties (flexural modulus). Cessation of ethanol consumption after bone injury along with ad libitum feeding of the liquid control diet brought about a normal outcome of bone repair.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akihiko, N., Kuroda, K. and Yamada, A. (1995) Induction of cytochrome P450-dependent monooxygenase in serum-free cultured Hep G2 cells. Biochemical Pharmacology 50, 14071412.[ISI][Medline]
Baran, D. T., Teitelbaum, S. L., Bergfeld, M. A., Parker, G., Cruvant, E. M. and Avioli, L. V. (1980) Effect of alcohol ingestion on bone and mineral metabolism in rats. American Journal of Physiology 238, E507E510.
Bikle, D. D. (1993) Alcohol-induced bone disease. World Review of Nutrition and Dietetics 73, 5379.[Medline]
Bikle, D. D., Genant, H. K., Cann, C., Recker, R. R., Halloran, B. P. and Strewler, G. J. (1985) Bone disease in alcohol abuse. Annals of Internal Medicine 103, 4248.[ISI][Medline]
Buhler, R., Lindros, K. O., Nordling, A., Johansson, I. and Ingelman-Sundberg, M. (1992) Zonation of cytochrome P450 isozyme expression and induction in rat liver. European Journal of Biochemistry 204, 407412.[Abstract]
Chakkalakal, D. A., Lippiello, L., Wilson, R. F., Shindell, R. and Connolly, J. F. (1990) Mineral and matrix contributions to rigidity in fracture healing. Journal of Biomechanics 23, 425434.[ISI][Medline]
Chakkalakal, D. A., Novak, J. R., Fritz, E. D., Mollner, T. J., Garvin, K. L. and McGuire, M. H. (1996) Osteoconductive and osteoinductive processes in bone healing. Transactions of the Orthopaedic Research Society 21, 586.
Chakkalakal, D. A., Strates, B. S., Mashoof, A. A., Garvin, K. L., Novak, J. R., Fritz, E. D., Mollner, T. J. and McGuire, M. H. (1999) Repair of segmental bone defects in the rat: an experimental model of human fracture healing. Bone 25, 321332.[ISI][Medline]
Chakkalakal, D. A., Strates, B. S., Garvin, K. L., Mollner, T. J., Novak, J. R., Fritz, E. D. and McGuire, M. H. (2000) Osteoinduction in a tissue scaffold during bone repair. Transactions of the Society for Physical Regulation in Biology and Medicine 19, 1213.
Chakkalakal, D. A., Strates, B. S., Garvin, K. L., Novak, J. R., Fritz, E. D., Mollner, T. J. and McGuire, M. H. (2001) Demineralized bone matrix as a biological scaffold for bone repair. Tissue Engineering 7, 161177.[ISI][Medline]
Chavassieux, P. S., Serre, C. M., Vernaud, P., Delmas, P. D. and Meuneir, P. J. (1993) In vitro evaluation of dose-dependent effects of ethanol on human osteoblastic cells. Bone and Mineral 22, 95103.[ISI][Medline]
Diamond, T., Stiel, D., Lunzer, M., Wilkinson, M. and Posen, S. (1989) Ethanol reduces bone formation and may cause osteoporosis. American Journal of Medicine 86, 282288.[ISI][Medline]
Diez, A., Serrano, S., Cucurull, J., Marinoso, L. L., Bosch, J., Puig, J., Nogues, X. and Aubia, J. (1997) Acute effects of ethanol on mineral metabolism and trabecular bone in SpragueDawley rats. Calcified Tissue International 61, 168171.[ISI][Medline]
Donohue, T. M., Jr, Sorrell, M. F. and Tuma, D. J. (1987) Hepatic protein synthetic activity in vivo after ethanol administration. Alcoholism: Clinical and Experimental Research 11, 8086.[ISI][Medline]
Dumbleton, J. H. and Black, J. (1981) Principles of mechanics. In Clinical Biomechanics, Black, J. and Dumbleton, J. H. eds, pp. 359400. Churchill Livingstone, New York.
Dyer, S. A., Buckendahl, P. and Sampson, H. W. (1998) Alcohol consumption inhibits osteoblastic cell proliferation and activity in vivo. Alcohol 16, 337341.[ISI][Medline]
Eriksson, C. J. P., Sippel, H. W. and Forsander, D. A. (1977) The determination of acetaldehyde in biological samples by head-space chromatography. Analytical Biochemistry 80, 116124.[ISI][Medline]
Eshbach, O. W. and Souders, M. (1975) Handbook of Engineering Fundamentals, 3rd edn, p. 450. Wiley, New York.
Folch, J., Lees, M. and Sloane-Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497509.
Friday, K. E. and Howard, G. A. (1991) Ethanol inhibits human bone cell proliferation and function in vitro. Metabolism 40, 562565.[ISI][Medline]
Garcia-Sanchez, A., Gonzalez-Calvin, J. L. and Diez-Ruiz, A. (1995) Effect of acute alcohol ingestion on mineral metabolism and osteoblastic function. Alcohol and Alcoholism 30, 449453.[Abstract]
Groves, W., Davis, J. F. C. and Sells, B. H. (1968) Spectrophotometric determination of microgram quantities of protein without nucleic acid interference. Analytical Biochemistry 22, 195210.[ISI][Medline]
Guzman, M. and Castro, J. (1990) Zonal heterogeneity of the effects of chronic ethanol feeding on hepatic fatty acid metabolism. Hepatology 12, 10981105.[ISI][Medline]
Hogan, H. A., Sampson, H. W., Cashier, E. and Ledoux, N. (1997) Alcohol consumption by young actively growing rats. A study of cortical bone histomorphometry and mechanical properties. Alcoholism: Clinical and Experimental Research 21, 809816.[ISI][Medline]
Hu, Y., Ingelman-Sundberg, M. and Lindros, K. O. (1995) Induction mechanisms of cytochrome P450 2E1 in liver: interplay between ethanol treatment and starvation. Biochemical Pharmacology 50, 155161.[ISI][Medline]
Janicke-Lorenz, J. and Lorenz, R. (1984) Alcoholism and fracture healing: a radiological study in the rat. Archives of Orthopaedic and Trauma Surgery 103, 286289.[ISI][Medline]
Klein, R. F. (1997) Alcohol-induced bone disease: impact of ethanol on osteoblast proliferation. Alcoholism: Clinical and Experimental Research 21, 392399.[ISI][Medline]
Koop, D. (1986) Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isoenzyme 3a. Molecular Pharmacology 29, 399404.[Abstract]
Kusy, R. P., Hirsch, P. F. and Peng, T.-C. (1989) Influence of ethanol on stiffness, toughness, and ductility of femurs of rats. Alcoholism: Clinical and Experimental Research 13, 185189.[ISI][Medline]
Lalor, B. C., France, M. W., Powell, D., Adams, P. H. and Counihan, T. B. (1986) Bone and mineral metabolism and chronic alcohol abuse. Quarterly Journal of Medicine, New Series 59, 497511.
Lieber, D. and DeCarli, M. (1982) The feeding of alcohol in liquid diets: two decades of application and a 1982 update. Alcoholism: Clinical and Experimental Research 6, 523531.[ISI][Medline]
Lindros, K. O. and Jarvelainen, H. A. (1998) A new oral carbohydrate alcohol liquid diet producing liver lesions: a preliminary account. Alcohol and Alcoholism 33, 347353.[Abstract]
Mueller, M., Schilling, T., Minne, H. W. and Ziegler, R. (1991) A systemic acceleratory phenomenon (SAP) accompanies the regional acceleratory phenomenon (RAP) during healing of a bone defect in the rat. Journal of Bone and Mineral Research 6, 401410.[ISI][Medline]
Nimni, M. E., Bernick, S., Cheung, D. T., Ertl, D. C., Nishimoto, S., Paule, W. J., Salka, C. and Strates, B. S. (1988) Biochemical differences between dystrophic calcification of cross-linked collagen implants and mineralization during bone induction. Calcified Tissue International 42, 313320.[ISI][Medline]
Nyquist, F., Berglund, M., Nilsson, B. E. and Obrant, K. J. (1997a) Nature and healing of tibial shaft fractures in alcohol abusers. Alcohol and Alcoholism 32, 9195.
Nyquist, F., Karlsson, M. K., Obrant, K. J. and Nilsson, J. A. (1997b) Osteopenia in alcoholics after tibia shaft fractures. Alcohol and Alcoholism 32, 599604.
Nyquist, F., Halvorsen, V., Madsen, J. E., Nordsletten, L. and Obrant, K. J. (1999) Ethanol and its effects on fracture healing and bone mass in male rats. Acta Orthopaedica Scandinavica 70, 212216.[ISI][Medline]
Passeri, L. A., Ellis, E. and Sinn, D. P. (1993) Relationship of substance abuse to complications with mandibular fractures. Journal of Oral and Maxillofacial Surgery 51, 2225.[ISI][Medline]
Peng, T.-C., Kusy, R. P., Hirsch, P. F. and Hagaman, J. R. (1988) Ethanol-induced changes in morphology and strength of femurs of rats. Alcoholism: Clinical and Experimental Research 12, 655659.[ISI][Medline]
Peris, P., Pares, A., Guanabens, N., Pons, F., De Osaba, M. J. M., Caballeria, J., Rodes, J. and Munoz-Gomez, J. (1992) Reduced spinal and femoral bone mass and deranged bone mineral metabolism in chronic alcoholics. Alcohol and Alcoholism 27, 619625.[Abstract]
Pierce, R. O. and Perry, A. (1991) The effect of ethanol on bone mineral. Journal of the National Medical Association 83, 505508.[ISI][Medline]
Sampson, H. W. (1997) Alcohol, osteoporosis, and bone regulating hormones. Alcoholism: Clinical and Experimental Research 21, 400403.[ISI][Medline]
Sampson, H. W., Chaffin, C., Lange, J. and DeFee, B. (1997) Alcohol consumption by young actively growing rats. A histomorphometric study of cancellous bone. Alcoholism: Clinical and Experimental Research 21, 352359.[ISI][Medline]
Sampson, H. W., Hebert, V. A., Boone, H. L. and Champney, T. H. (1998) Effect of alcohol consumption on adult and aged bone: composition, morphology, and hormone levels of a rat animal model. Alcoholism: Clinical and Experimental Research 22, 17461753.[ISI][Medline]
Shaffer, J. P. (1986) Modified sequentially rejective multiple test procedures. Journal of the American Statistical Association 81, 826831.[ISI]
Strates, B. S. and Tiedeman, J. J. (1993) Contribution of osteoinductive and osteoconductive properties of demineralized bone matrix to skeletal repair. European Journal of Experimental and Musculoskeletal Research 2, 6167.
Tennesen, H., Pedersen, A., Jensen, M. R., Moller, A. and Madsen, J. C. (1991) Ankle fractures and alcoholism. The influence of alcoholism on morbidity after malleolar fractures. Journal of Bone and Joint Surgery 73B, 511513.[ISI]
Torzilli, P. A., Burstein, A. H., Takebe, K., Zika, J. C. and Heiple, K. G. (1981) The material and structural properties of maturing bone. In Mechanical Properties of Bone, Cowin, S. C. ed., pp. 145161. American Society of Mechanical Engineers, New York.
Tsukamoto, H., French, S. W. and Benson, N. (1985a) Severe and progressive steatosis and focal necrosis in rat liver induced by continuous intragastric infusion of ethanol and low fat diet. Hepatology 5, 224232.
Tsukamoto, H. F., French, S. W., Reidelberger, R. D. and Langman, C. (1985b) Cyclical pattern of blood ethanol levels during continuous intragastric infusion of rats. Alcoholism: Clinical and Experimental Research 9, 3137.
Turner, R. T., Greene, V. S. and Bell, N. H. (1987) Demonstration that ethanol inhibits bone matrix synthesis and mineralization in the rat. Journal of Bone and Mineral Research 2, 6166.[ISI][Medline]
Turner, R. T., Spector, M. and Bell, N. H. (1991) Ethanol induced abnormalities in bone formation, turnover, mechanical properties and mineralization in young adult rats. Cells and Materials 1, (Suppl.), 167173.
Wu, D. and Cederbaum, A. I. (1996) Expression of cytochrome P4502E1 in rat fetal hepatocyte culture. Molecular Pharmacology 49, 802807.[Abstract]