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- MATERIALS AND METHODS
We characterized the composition and mechanical properties of cortical bone during maturation and growth and in adult life in the rabbit. We hypothesized that the collagen network develops earlier than the mineralized matrix. Growth was monitored, and the rabbits were euthanized at birth (newborn), and at 1, 3, 6, 9, and 18 months of age. The collagen network was assessed biochemically (collagen content, enzymatic and non-enzymatic cross-links) in specimens from the mid-diaphysis of the tibia and femur and biomechanically (tensile testing) from decalcified whole tibia specimens. The mineralized matrix was analyzed using pQCT and 3-point bend tests from intact femur specimens. The collagen content and the Young's modulus of the collagen matrix increased significantly until the rabbits were 3 months old, and thereafter remained stable. The amount of HP and LP collagen cross-links increased continuously from newborn to 18 months of age, whereas PEN cross-links increased after 6 months of age. Bone mineral density and the Young's modulus of the mineralized bone increased until the rabbits were at least 6 months old. We concluded that substantial changes take place during the normal process of development in both the biochemical and biomechanical properties of rabbit cortical bone. In cortical bone, the collagen network reaches its mature composition and mechanical strength prior to the mineralized matrix. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1626–1633, 2010
Collagen is the principal structural component of bone matrix, accounting for about 1/3 of mineralized bone tissues.1 During development, bone formation and growth rates are regulated by genetic and hormonal factors, and by external factors such as mechanical loading that interact to determine the final anatomy and strength of the skeleton.1–3 During this period, bone also changes in its composition, structure, and function. The organic matrix changes its composition and structural organization, and mechanical stiffness increases with increased bone mineral density (BMD).3, 4 The majority (>80%) of the mineral can be found within the collagen fibril network,5 but limited information is available about the factors that determine the normal deposition of mineral, for example, the timing and how this can determine the properties of the adult tissue.
The structure and function of maturing and growing bone were studied in several animal models.4, 6, 7 Most studies focused on bone development and growth in rabbits, investigating the development of the primary and secondary ossification centers or evaluating bone growth at the growth plate.8–10 Other studies showed that BMD increases and biomechanical properties change concurrently during maturation and growth.4, 6 However, the development of the collagen framework has been less extensively studied. During development, at least in the equine, the collagen matrix is formed rapidly, and the fibrils are restructured and re-orientated with time towards the main loading directions of the bone.4 In addition, changes also occur in the amounts of intra- and interfibrillar enzymatic (hydroxylysylpyridinoline, HP; lysylpyridinoline, LP) and non-enzymatic (pentosidine, PEN) cross-links.11–14 In experimental studies, the collagen framework in long bones of mice and rats undergoes significant structural modifications during growth and maturation.6, 15, 16 A previous study in mice found that the content and mechanical strength of the collagen matrix increased during early maturation,6 whereas during adult age, the collagen composition remained stable despite decreased stiffness of the collagen matrix.17 Also, in equine long bones, collagen orientation alters significantly during maturation and growth towards a more longitudinal fibril direction.18 Moreover, studies on equine subchondral bone showed that both the biochemical and structural modifications of the collagen network primarily develop during the first half year post-partum.4, 19 Comparison between studies is often difficult due to differences in species, anatomical location, type of bone (e.g., cortical, trabecular, or subchondral bone), and experimental conditions. Furthermore, how the properties of the collagen matrix, the mineral content, and the mechanical integrity of bone develop in relation to one another, and how they individually and together control the bone's mechanical resistance to fracture remains unknown.
We aimed to characterize the development of the collagen network, its cross-links, and its relationships to the mechanical properties in demineralized cortical bone of the rabbit. Simultaneously, these characteristics were evaluated in relation to quantified properties of the mineralized bone. Hence, we aimed to clarify the contributions of the mineral content and collagen matrix to the functional properties and to reveal the timing of the development of the collagen network and its relation to the mineralized matrix properties. We hypothesized that the development of the collagen network in bone precedes the progress of skeletal calcification and that the mechanical strength of the network reaches its mature levels prior to the mineralized matrix. The biochemical and mechanical development of the collagen and mineralized matrix in rabbit cortical bone was assessed from birth to mature adult age.
- Top of page
- MATERIALS AND METHODS
We found significant changes in the biochemical and mechanical properties of bone during maturation and growth of female rabbits. Some changes occurred over the lifetime, such as the increase in BMD and non-enzymatic PEN cross-links, but the most significant changes took place before the bone tissue reached maturity. Collagen content increased up to 3 months of age. Most mechanical characteristics of the collagen network also reached their mature properties at about 3 months. However, the force needed to break the collagen network increased further until 6 months, most likely due to continued bone growth. This coincided with a continued increase in the numbers of collagen cross-links, indicating that the cross-links augment the stability and mechanical strength of the collagen network. Conversely, mineralized bone tissue demonstrated continued mineral deposition, increase in cortical thickness, and enhanced bone mechanical properties up to at least 6 months of age (Fig. 7). Knott and Bailey12 proposed that the mineralization status of bone depends on the maturation of the collagen network and that post-translational modifications of the network play a key role in mineralization. Wassen et al.5 demonstrated that the collagen network structure directs the mineralization process. Others have proposed that mineralization prevents further cross-linking.28, 29 These findings are consistent with our results, where the mineralization followed the development of the collagen network and continued to develop even after the collagen content reached its mature levels. Our study clearly indicates that the compositional and mechanical properties of bone change most during the early dynamic and rapid period of growth, that is, the first 3–6 months of life in the rabbit. Weight gain and increase of physical activity are most likely stimuli that determine this process. Withholding physical activity during early life may affect the development process, and hence, the biochemical composition of the bone matrix.6, 19
Figure 7. Comparison between the development of the collagen content and the mineral density in the mid-diaphysis of the femur showed that the collagen content reached its maximal levels at 3 months of age, whereas mineral content continued to increase at least until 6 months. Results are expressed as mean ± SD (*p < 0.05, **p < 0.001).
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We investigated bone properties in both the femur and tibia. Timing of the development of certain properties can be different in these two long bones. For example, studies of the growth plates in rabbits showed that the bone growth rate in the tibia is higher.8 Furthermore, articular cartilage in the rabbit tibia reaches its mature composition and structure before femoral cartilage.30 However, we measured both BMD and collagen content at the same location (mid-diaphysis of the femur). The comparison was based on different, but standard techniques. Mineral content measured by pQCT correlates closely with mineral content based on non-destructive neutron activation analysis and flame atomic absorption spectrometry,31 and with ash weight.32 Nonetheless, comparison between these techniques must be done with care. Therefore, only a comparison of the trends with age was conducted, showing that collagen content reached its maximal levels before the mineralized matrix (Fig. 7).
Bone mineral content correlated with the weights of the animals (Fig. 6a). Previous studies showed linear correlations between these parameters,6, 33 but did not include newborn animals. The energy needed to tear the decalcified collagen network of the tibia was about two times higher than that measured from the 3-point bend tests of intact bone (Fig. 6b). This confirms previous studies suggesting that the collagen network is responsible for storing energy in the bone and that the mineralized matrix decreases the capability to store energy.3, 34 We found a relationship between the collagen content and the modulus of the decalcified tibia bone matrix (Fig. 6c). Moreover, the amount of enzymatic cross-links, HP and LP, correlated with ultimate strain (Fig. 6d). This supports earlier findings in mice, which indicated that the collagen network's ultimate displacement was associated with the amount of LP cross-links.17 However, we did not determine other cross-links in connective tissues, such as pyrrolic cross-links, MODIC, GODIC, DOGDIC, or glucosepane.11, 35, 36
A limitation of our study is that due to the highly varying size of the specimens, all variables could not be kept constant between age groups during mechanical testing (Fig. 1). However, the parameters were set to achieve similar relative rates and span length for each age group and specimen. We believe this is also the strength of our study, since we know of no previous study in which mechanical and compositional properties were measured over the entire life span of the rabbit. Furthermore, mechanical testing was conducted at room temperature. The mechanical properties of bone are influenced by temperature; for accurate measurements, specimens should be tested at 37°C. However, this is not always practical. Previous studies showed that testing at room temperature increased the modulus of bone by about 2–4% compared to testing at 37°C.26 Thus, the error caused by testing at room temperature is not large, and any temperature dependence would be predicted to influence each experimental group equally.
Previous studies in mice described comparable compositional and mechanical developmental curves.6, 17 However, those studies did not include newborns. Studies on equine bone showed that the biochemical and structural modifications of the collagen network primarily take place during the first 6 months post-partum.4, 18, 19 Accounting for variation in timing of skeletal maturity, our results generally agree with these previous findings. However, due to the choice of experimental time points in the previous studies, the time points when the collagen and mineral content reached their respective mature levels were not established.
All rabbits used in our study were females. Evidence exists that gender affects cortical bone growth rate and adaptation to exercise.37 However, we cannot draw any conclusions on gender effects. The rabbits in our study become skeletally mature between 6–9 months of age, in line with a recent report that demonstrated negligible bone growth in rabbits after 6–8 months, while articular cartilage showed mature structure by 3–4 months.38 It was evident that the bone's capacity to store energy had decreased at 18 months (Figs. 4c and 5c). This may be due to decreased viscoelasticity of the collagen matrix with age,34 which may be related to an increase in PEN concentration.12, 14, 17 Due to the non-enzymatic character, PEN cross-links reflect the relative tissue age and matrix turnover. The decrease of PEN concentration during the early time points, expressed per collagen, was most likely due to the rapid increase in collagen content during early age. With age PEN cross-links increase as synthesis and turnover of collagen are reduced. When the animals get older, the cross-links increase because collagen turnover is low. This is supported by our findings (Fig. 2d) and in earlier studies in mice.6, 17
We conclude that profound biochemical and biomechanical changes take place during the early process of maturation and growth in both the mineralized and collagen matrix of rabbit cortical bone. Our study demonstrates that collagen content and biomechanical characteristics of the collagen network reached a mature state at or after 3 months of age in the rabbit, whereas the mineralized matrix reached its mature levels at or after 6 months of age.