Age-Related Effects of Advanced Glycation End Products (Ages) in Bone Matrix on Osteoclastic Resorption
Yang X1,3 • Gandhi C 2 • Rahman MM4 • Appleford M 2 • Sun LW 1 • Wang X2,3
1 School of Biological Sciences and Medical Engineering, Beihang University, Beijing, China
2 Departments of Biomedical Engineering, The University of Texas at San Antonio (UTSA), One UTSA Circle, San Antonio, TX 78249, USA
3 Department of Mechanical Engineering, The University of Texas at San Antonio (UTSA), One UTSA Circle, San Antonio, TX 78249, USA
4 Department of Medicine, University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, USA
Advanced glycation end products (AGEs) accumulate in bone extracellular matrix as people age. Previous studies have shown controversial results regarding the role of in situ AGEs accumulation in osteoclastic resorption. To address this issue, this study cultured human osteoclast cells directly on human cadaveric bone slices from different age groups (young and elderly) to warrant its relevance to in vivo conditions. The cell culture was terminated on the 3rd, 7th, and 10th day, respectively, to assess temporal changes in the number of differentiated osteoclasts, the number and size of osteoclastic resorption pits, the amount of bone resorbed, as well as the amount of matrix AGEs released in the medium by resorption. In addition, the in situ concentration of matrix AGEs at each resorption pit was also estimated based on its AGEs autofluorescent intensity. The results indicated that (1) osteoclastic resorption activities were significantly correlated with the donor age, showing larger but shallower resorption pits on the elderly bone substrates than on the younger ones; (2) osteoclast resorption activities were not significantly dependent on the in situ AGEs concentration in bone matrix, and (3) a correlation was observed between osteoclast activities and the concentration of AGEs released by the resorption. These results suggest that osteoclasts tend to migrate away from initial anchoring sites on elderly bone substrate during resorption compared to younger bone substrates. However, such behavior is not directly related to the in situ concentration of AGEs in bone matrix at the resorption sites.
KEYWORDS: Advanced glycation end products (AGEs); Bone; Bone resorption; Osteoclast
Advanced glycation end-products (AGEs) are products of non-enzymatic glycation of long-lived proteins (e.g. collagen) and lipids . AGEs may accumulate in the body as people age and may be accelerated in diabetes mellitus patients with poorly controlled hyperglycemia . Such accumulation of AGEs may cause many clinical complications [3-7], including delayed bone healing and diabetic osteoporosis. The effects of AGEs on bone are manifested in two aspects: one is the deleterious modification of bone ultrastructure (e.g. non-enzymatic cross linking in collagen), which may directly lead to the reduced toughness of bone [2, 8-10]. The other is the negative modulation of cellular activities in bone remodeling process , which include abnormal resorption activity of osteoclasts [11-17]. However, the underlying mechanisms are still poorly understood.
It is not clear whether or not osteoclasts would react differently with the bone matrix that has distinct AGEs concentration. In addition, it is not clear whether or not the AGEs in bone matrix released into the serum by osteoclast resorption would affect the cell function and activities of the osteoclasts. Currently, not evidence shows that anchoring of osteoclasts on bone surfaces is affected by the in situ concentration of AGEs in bone matrix, while it has been reported that a high concentration of AGEs in serum always imposes negative impacts to bone cell activities. So, we hypothesized that the osteoclast resorption is not directly dependent on the in situ concentration of matrix AGEs, but correlated the amount of matrix AGEs released to serum by resorption, which is increased with age.
First, we examined whether or not osteoclasts preferred to anchor on the bone surface that has high in situ concentrations of matrix AGEs. To do so, we measured the in situ AGEs concentration of each resorption pit and then obtained the histogram of number of resorption pits with respect to the in situ AGEs concentration at the pits. The histogram demonstrated that osteoclasts anchored on the bone surface independent of the in situ AGEs concentration. When we examined the correlation between the total areas of the resorption pits and the bone surface, we found these two were a strongly linearly correlated. Similarly, a linear correlation existed between the number of resorption pits and the actual bone area. All the above results indicate that the osteoclasts activities are not directly related to the in situ AGEs within the bone matrix. Then, the next question is that how matrix AGEs affect the activities of osteoclasts if the AGEs do not have a direct impact on the anchoring of osteoclasts.
Then, it is noteworthy from our study that the concentration of AGEs in bone matrix was much higher in elderly bone than young bone and the overall resorption activities of osteoclasts were significantly altered as the donor age increases. There observations imply that age-related accumulation of AGEs in bone matrix may have an indirect effect on the osteoclast activities. One possible pathway is that in situ AGEs are released from bone matrix by osteoclast resorption, and the released AGEs in turn make the local concentration of AGEs in serum elevated. Finally, the much higher concentration of AGEs in serum may be a major cause of altered osteoclast activities on elderly bone substrates.
To test the hypothesis, we used the amount of calcium released into serum during cell cultures as a measure of total tissue volume resorbed from bone by osteoclasts (assuming little changes in bone density). Then, we estimated the amount of AGEs released into the serum as the product of total resorbed tissue volume and the AGEs concentration in the tissue. The results showed that the amount of released AGEs in the serum increased with the donor age and such changes were significantly correlated with the osteoclastic resorption activities. These above results obtained suggest that the elevated concentration of AGEs due to matrix AGEs released from bone matrix into the local environment via the resorption process is most likely the reason for age-related changes in the osteoclasts activities.
Another interesting finding of this study is that osteoclast resorption patterns are significantly correlated with the donor age of bone substrates, showing much larger but shallower resorption cavities in elderly group compared to the younger ones. Since the in situ concentration of matrix AGEs is much higher in elderly bone substrate, this observation suggests that osteoclasts are not very happy at the initial anchoring location and tend to migrate away when the in situ concentration of matrix AGEs are higher. It is possible that the AGEs released by osteoclasts in the resorption capsule may have triggered the cell migration from the initial anchoring location.In summary, the underlying mechanism of the effect of matrix AGEs on osteoclast activities may be depicted in Fig. 1. First, osteoclast anchoring is not directly affected the in situ concentration of matrix AGEs in bone. However, the released AGEs from bone matrix make osteoclasts unhappy and tend to migrate away from the initial anchoring location. Then, the soluble AGEs released into serum by resorption may affect differentiation and activation of osteoclasts.
Importance of the study: we demonstrate in this study that aging may alter the activities of osteoclasts in terms of differentiation/activation and resorption (i.e. larger but shallower resorption cavities on elderly bone substrates compared to younger ones). the age-related changes in the osteoclast resorption activities are most likely due to the elevated amount of matrix AGEs released into serum by osteoclast resorption. These new findings are important to understand the underlying mechanism of AGEs in affecting bone quality and to help develop effective therapeutical strategies to prevent the adverse effects of AGEs on skeletal tissues.
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