Creatine supplementation research has focused primarily on its effects on skeletal muscle. Some effects of creatine monohydrate (CM) supplementation on skeletal muscle and exercise performance include increased muscle power (29), increased fat-free mass (FFM) in healthy younger (17) and older adults (4), and increased strength in healthy individuals (32) and in patients with a variety of neuromuscular disorders (27). Physiologically, creatine is used predominantly by tissues with high energy demands. Tissues that have been found to contain high levels of creatine kinase (CK) activity include skeletal and cardiac muscle, brain, and spermatozoa (33). It has been noted recently that CK activity is also required for the formation of endochondral bone (9) and is a pivotal enzyme in the cellular energy metabolism of osteoblasts. Further corroborating evidence for the importance of CK in skeleton formation is that high CK activity is noted in chondrocytes (13). Creatine kinase-B expression is upregulated during periods of increased energy demands in osteoblasts (26), allowing for changes in cell shape and adherence of osteoblast cells to bone surfaces (5). CK plays a role in the cytoskeleton and in increased activity of the membrane pumps, which, in turn, are related to the formation of extracellular matrix and mineralization in differentiated osteoblasts (5).
Bone growth, modeling, and remodeling are highly energy-dependent processes (8) and can be influenced by nutrient availability, mechanical loads (20), and drugs (bisphosphonates) (6). A recent study by Gerber et al. (10) has demonstrated that supplementing low-serum cell culture medium with pure creatine has a stimulatory effect on rat osteoblastlike cell metabolic activity, differentiation, and mineralization. Gonadal steroids also contribute to the stimulation of bone growth and maintenance of balanced bone-turnover rates, with CK being directly involved in those processes (33). Moreover, gonadal steroids and vitamin D can enhance the differentiation of CK-B (25,26,33), and osteoblast CK activity is an indicator of the efficacy of interventions designed to prevent osteoporosis (25). To date, hormones such as vitamin D, 17-β-estradiol, testosterone, and parathyroid hormone have been observed to stimulate CK activity in bone tissue (24,25). Despite the importance of CK in bone formation, there has been limited research examining the effects of supplemental CM on bone metabolism.
Recent clinical evidence suggests that CM supplementation may have positive alterations on bone metabolism. An increase in the bone mineral density (BMD) and a decrease in the N-telopeptides were observed after supplementation with CM in boys with Duchenne (DMD) and Becker's muscular dystrophy (14). We have recently confirmed the observation of reduced urinary N-telopeptide excretion after CM supplementation in boys with DMD (30). These observations suggest that CM has the potential to favorably influence bone metabolism. In the latter study (30), the effects of creatine on reduced N-telopeptide excretion were independent of corticosteroid use; this has relevance to other medical conditions for which corticosteroids are used (e.g., asthma and chronic obstructive pulmonary disease) with negative side effects on bone. The objective of this study was to determine the influence of supplementation with CM on bone structure and function in growing rats, to establish a therapeutic model. On the basis of the current evidence, we hypothesized that CM supplementation would positively affect bone function and structure. Because of our interest in pediatric neuromuscular conditions where corticosteroids are used for therapy (30), and because osteopenia is seen in 46% of children using high-dose inhaled steroids (7), we chose to evaluate the efficacy of creatine supplementation on bone structure and function in young, growing rats.
MATERIAL AND METHODS
Animals and housing.
Thirty-two male Sprague-Dawley rats (Taconic Laboratories, Germantown, NY) were separated into two dietary groups. The rats were 5 wk old at the commencement of the study and were Pneumocystis carinii free. The experiment lasted for a period of 8 wk. The protocols were approved by the McMaster University animal review and ethics board and conformed to all guidelines of the Canadian Council on Animal Care.
Animals were housed in pairs in microisolator cages within the McMaster University central animal facility. Ear clippings were used to distinguish cage mates. The animals were maintained on a 12:12 light-dark cycle at approximately 22°C. The rats were acclimatized to the facility, cages, and a standard rodent diet (2016 Global Rodent Diet, Harlan-Teklad, Madison, WI) for 2 wk before initiation of the experimental interventions. The rats also had ad libitum access to food and water for the duration of the study.
At the onset, animals were randomized into one of two experimental groups: 1)control group (CON), or 2) creatine group (CR) (2% CM w.w.), ensuring that the mean body mass at the onset of the experimental group was identical for each group.
The experimental diet was a manipulation of the standard rodent diet and was custom manufactured by a commercial supplier (Harlan-Teklad, Madison, WI) by combining CM (99.9% CM, as determined by high-performance liquid chromatography; Creapure, Traco Labs Inc., Champaign, IL) with ground 2016 Global rodent diet, which was then repelleted.
On completion of the 8-wk intervention, the rats were euthanized with an overdose of sodium pentobarbital, and the femurs were removed. One femur was wrapped in gauze saturated with saline and was stored (−80°C) for later mechanical analyses.
Assessment of body composition and bone density.
All animals underwent a dual-energy x-ray absorptiometry (DEXA) scan at two time points: before commencement of dietary supplementation, and on the day preceding the completion of the study. To complete the DEXA scans, each animal was administered a light anesthetic (isoflurane) and placed on the scanning bed, and three scans were performed: one of the whole body, one of the femur, and one of the L5 lumbar vertebra. The whole-body scan was used to determine body composition (i.e., % fat and lean mass), total bone mineral content (BMC), and total BMD. The femoral and vertebral scans provided regional measures of BMC and BMD. All scans were performed using a QDR-4500 fan beam scanner equipped with small-animal software (Hologic, Inc., Bedford, MA).
Bone mechanical properties.
To measure the effects of creatine on the mechanical parameters of bone, a materials-testing system (Model 4442, Instron, Burlington, ON) coupled to a computer was used to test the femurs in three-point bending. Of the 32 femurs collected for fracture studies, the first 20 were kept frozen at −80°C. The day before mechanical testing, the frozen (−80°C) femurs were placed in a 0.1 M phosphate-buffered saline solution to thaw overnight at 4°C. Femurs from the remaining 12 rats (six each from the CON and CR groups) were subjected to mechanical testing within 8 h of sacrifice (maintained at 4°C). A previous study by another lab group found that there were no significant effects of freezing on bone mechanical properties (19), and equal numbers of treated and nontreated rats were studied under each condition.
The three-point bending mechanical test involved the femurs being placed on two lower supports 15.9 mm apart, with the anterior surface facing upwards. Each femur was loaded at the middiaphysis on the anterior side at a rate of 0.17 mm·s−1 (10 mm·min−1) until fracture. Structural properties (maximal load, load to failure, and energy to failure) were calculated from the generated force-displacement data using automated materials-testing software (Merlin series IX, V. 7.51) (23). After the femurs were fractured, the diameter of each failure site was measured, using a handheld caliper measurement system.
Data were evaluated using a one-way independent analysis of variance (ANOVA), with diet as the main variable to compare the change in bone density and content as well as body composition during 8 wk. Independent t-tests were used to determine significance among the remaining compression variables. All data were analyzed at a significance level of P = 0.05 (STATISTICA 5.1, Statsoft, Tulsa, OK), and all data are expressed as a means ± SD.
Bone mineral and body composition analysis.
Lumbar BMD in the CR rats was significantly greater (P < 0.05) than in the age-matched controls after 8 wk of supplementation (Fig. 1). Distal femoral BMD in the CR rats also tended to be greater than in the CON rats, but the difference was not significant (P = 0.06). No significant differences were observed in BMC changes (Fig. 2) or body composition (Fig. 3) for the two different diets.
Bone mechanical properties.
The load to failure for femurs from CR rats was significantly greater (+12.3%) than for femurs from age-matched control rats (Table 1). Also, the diameter of the failure site on the middiaphysis of the CR rat femurs was significantly larger than those from the CON group (P < 0.05). No significant differences were observed for measurements of maximal load or energy at failure.
We found that dietary supplementation with CM in young, growing rats resulted in a significant increase in BMD of the L5 lumbar vertebrae, with a concomitant trend towards an increased BMD in the distal femur. It was also found that the CR group withstood a greater maximal load. Also, the diameter of the femurs at the failure site was significantly larger in the CR rats (P < 0.05). We have previously demonstrated that the growth properties of the animals in the two different dietary groups was the same (21).
Both osteoblasts and chondrocytes are dependent on creatine kinase for energy and are important in skeleton formation (5,13,26); thus, the bone metabolism in these growing rats may have been favorably altered with dietary supplementation of CM. This idea is further supported by Gerber et al. (10), who have demonstrated recently that supplementation with pure creatine to rat osteoblastlike cells has the capacity to alter the cell's metabolic activity. Because trabecular bone is more abundant in the vertebrae than in the femur, the significant increase observed in BMD in the vertebrae over the femur likely reflects the vertebral bone as a more sensitive anatomic area, because it has a greater region of osteoblast activity and bone turnover. Because of the abundant regions of bone turnover in the vertebrae, the increase in BMD may signify that creatine altered trabecular bone metabolism in this region. However, further analyses of the remodeling sites in this area are required before any conclusions can be drawn. Changes in BMC were not statistically significant, but trends were similar to the changes observed for BMD.
One possible explanation for the positive results seen in the creatine-supplemented group is that creatine may have influenced the bone microarchitecture, although this postulation cannot be corroborated until microarchitecture attributes, such as trabecular number, trabecular thickness, etc., are examined. It may be that creatine influenced the development of trabeculae during the endochondral bone formation through the hypertrophic chondrocytes. Alternately, creatine may influence the activation of more bone-modeling sites or increase the osteoblasts' ability to lay down increased amounts of bone. Recently, a stimulatory effect of creatine supplementation on metabolic activity, differentiation, and mineralization in rat osteoblastlike cells in culture has been observed (10). To determine whether the above-mentioned hypotheses and findings occur at the tissue level, further examination of the bone microarchitecture and osteoblast activity, using histomorphometric analysis and mineral apposition rates of the vertebrae and femur, is needed.
An important factor in considering bone strength, apart from material properties and microarchitecture, is the bones' size and/or geometry. At the periosteal surface, cellular events are responsible for bone diameter (18). Because the maximal load (P < 0.05) and the diameter of the bone at the fracture site (P < 0.05) were increased in CR animals, one possible explanation for the observed increases may be that creatine was able to alter the periosteal apposition in the femurs. The effect of CM supplementation on periosteal remodeling events is not known, and further investigation is warranted to determine whether this is the case.
Research investigating the role of diet on bone has predominately focused on the effect of calcium (3,16), protein (35), fat and sucrose (34), parathyroid hormone (22), vitamin D (11), and other dietary minerals. To date, however, there has been limited research on the influence of relatively novel (yet widely used) dietary supplements, and their effects on bone growth and microarchitecture. Furthermore, the majority of therapeutic intervention studies involving bone have focused on pharmacological interventions (1). On the basis of such studies, it seems that the most potent drugs that influence bone metabolism and growth are the bisphosphonates (6). The suggested mechanism of action is the inhibition of bone resorption, ultimately resulting in a decreased rate of bone turnover and increased BMC (6). Thus, the focus of most of the ongoing research has been directed toward the prevention of bone loss. Despite the beneficial effects of bisphosphonates on bone, there is the potential for serious side effects (15), especially in children (2). In contrast, there have been very few substantiated side effects with the dietary CM supplementation in short- or intermediate-term human clinical studies or in therapeutic studies in animals (12,28). Furthermore, a recent histological study evaluating the effects of supplemental creatine in rats found no evidence of damage or inflammation in any tissues or organs (28). To date, only anecdotal evidence of possible negative side effects has been reported (31). On the basis of the current investigation, CM may represent a low-toxicity agent that could enhance BMD.
Dietary supplementation with CM also has been associated with alterations in lean mass. An increase in FFM is often observed with creatine supplementation in humans (4,17). The current investigation found that the CR rats tended to be slightly leaner, with marginally less total fat than the CON group (Fig. 2). Thus, the trend toward a change in the femoral BMC was not likely caused by a creatine-dependent increase in FFM in the muscle and increased force exertion on the bone; it was more likely attributable to the direct action of creatine on bone cell metabolism and/or function, particularly given the positive effect on lumber BMC.
Other observations in humans have suggested a possible beneficial role for dietary supplementation with creatine on bone metabolism. Louis et al. (14) observed increased BMD in young boys diagnosed with Duchenne and Becker muscular dystrophy when they were supplemented with CM for 3 months. The effect of creatine seemed to involve a reduction in bone breakdown, because they observed significantly less urinary N-telopeptide excretion (14). Furthermore, a recent study (30) has confirmed the finding of reduced urinary N-telopeptide excretion in a larger cohort of boys with Duchenne muscular dystrophy during a 4-month period. Taken together, those latter observations, the study by Gerber et al. (10), and the findings of the current investigation suggest that creatine supplementation may be beneficial as a therapeutic agent against disorders associated with decreased BMD in young, growing populations.
In summary, the current investigation has demonstrated that dietary supplementation with CM resulted in increased lumbar BMD and an increased failure load for cortical bone in the femoral middiaphysis. The dietary supplementation with creatine also had beneficial affects on rat femoral BMD. Further investigation is warranted to examine the mechanisms through which dietary creatine may influence the metabolic activity of osteoblasts, osteoclasts, and chondrocytes. The current data suggest that creatine supplementation has the potential to beneficially affect bone in young, growing rats and, therefore, may represent a novel intervention for disorders that adversely affect BMC. Future studies are required to confirm the current findings, to evaluate the potential efficacy of the intervention in other animal models of bone loss, and to further elucidate a possible mechanism.
We thank Ryan Simon for his assistance in performing mechanical loading data retrieval and technical support. Also, a special thank you to Kathleen R. Young for her research assistance while maintaining the 8-wk protocol.
This study was funded through a grant from the Hamilton Health Sciences Foundation.
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