Focal Therapeutic Irradiation Induces an Early Transient Increase in Bone Glycation

Abstract

Advanced glycation end products (AGEs) are an abnormal modification of the collagenous matrix in bone, and their accumulation contributes to alteration of mechanical properties. Using a mouse model of focal external radiotherapy, we quantified the time-dependent changes in the glycation of bone collagen following unilateral hindlimb exposure to 4 daily fractions of 5 Gy. Fluorometric analysis of decalcified femurs demonstrated a significant and transient increase in the quantity of pentosidine, pyridinolines, and non-specific AGEs per unit of collagen at one week post-radiation. These differences did not persist at 4, 8, 12, or 26 weeks post-radiation. Radiation had no effect on bone collagen content. We hypothesize that following the transient increase in glycation products, these cross-links are then removed as a result of increased post-radiation osteoclast activity and continued mineralization of the bone.

INTRODUCTION

Although fragility fractures are a frequent complication associated with radiation therapy for soft tissue sarcoma, there are currently no viable prevention strategies. Mechanical testing and computational modeling indicate that decreased strength in irradiated bone cannot be explained by changes in mineral density or bone quantity alone but are also likely due to embrittlement of the bone tissue (1). There is substantial laboratory and epidemiological evidence for the role of material properties in regulation of bone mechanical properties, including increased fragility associated with diabetes and age (2–20).

The organic matrix, consisting primarily of type I collagen, contributes extensively to the mechanical integrity of bone (20). Modification of collagen can occur through glycosylation, cross-linking, and fibril fragmentation (3, 21). Enzymatic cross-linking of collagen is typically biologically regulated and indicative of tissue maturity, while non-enzymatic cross-links are influenced by the local microenvironment and closely associated with aging and disease processes (16, 20). One factor contributing to altered matrix mechanical properties may be the accumulation of advanced glycation end products (AGEs). AGEs are naturally occurring non-enzymatic modifications of proteins. Matrix cross-links resulting from formation of AGEs can alter matrix integrity, cell-matrix signaling, and rates of tissue turnover (16, 17, 22–24).

We hypothesize that radiotherapy induces increased accumulation of AGEs in bone. Modification of bone collagen by accumulation of AGEs would provide a potential explanation for the altered material properties of bone post-radiation. The aim of this study was to quantify accumulation of advanced glycation end products in bone following radiotherapy using a mouse model.

METHODS

Hindlimb Irradiation

Female BALB/F mice aged 12 weeks (Taconic, Germantown, NY) were anesthetized and their hindlimbs extended for unilateral localized irradiation (RTx) at a dose of 20 Gy delivered as four consecutive daily 5 Gy fractions (4×5 Gy) (n=6/group/time point). The body and contralateral hindlimb of each animal was covered with lead shielding, allowing the non-irradiated hindlimb to serve as an internal non-irradiated control (0 Gy). The BED (Biologically Effective Dose) was calculated to be 57.5 Gy_2.8, with an EQD_{2 Gy} (Equivalent Dose in 2 Gy Fractions) of 32.5 Gy_2.8/2 (or ~16 fractions of 2 Gy each) where:

$$\mathit{BED}=nd\left(1+\frac{d}{\alpha /\beta }\right)$$

and

$${\mathit{EQD}}_2=\frac{\mathit{BED}}{\left(1+\frac{2}{\alpha /\beta }\right)}$$

In this case, n is the number of fractions, d is the dose per fraction (here, d = 5 Gy), and α/β = 2.8 Gy for bone, which is considered a normal and therefore late-responding (non-tumor) tissue (25–27). All methods were approved by the SUNY Upstate Medical University Committee for the Humane Use of Animals.

Analysis of Cross-Links

Tibias were harvested at 1, 2, 4, 8, and 26 weeks post-RTx, cleaned of soft tissues, and wrapped in saline-soaked gauze for storage at −20°C until processing. The tibias were then decalcified with 30% formic acid in 20% aqueous sodium citrate for 24 hours (Cat #399388 and #W302600, Sigma-Aldrich, St. Louis, MO, USA) and lyophilized (17). This was followed by digestion in collagenase Type II (3 mg/ml at 37°C, Cat #LS004176, Worthington Biochemical, Lakewood, NJ, USA) with homogenization (Polytron Kinematica, Lucerne, Switzerland). Insoluble organic material was removed by centrifugation. The extent of collagen cross-linking was determined by autofluorescence of the soluble matrix digest (Tecan Infinite M200, Research Triangle Park, NC). All samples were run in triplicate and four fluorescence reads were done per well. Three glycation products were quantified, including pentosidine (excitation/emission λ 335/385), pyridinolines (297/395), and non-specific AGEs (370/440 and 335/400). Pentosidine was quantified by comparison to a quinine sulfate standard curve (17). Results are expressed as arbitrary units (AU, arbitrary fluorescence units as reported by the plate reader using a gain setting of 100), with the exception of pentosidine, which is expressed in relation to the quinine sulfate standard. Fluorescence values were normalized by the moles of collagen contained in the same sample volume. The resulting value reflects the degree of cross-linking per collagen molecule, not the total number of cross-links in each bone sample. The collagen content of each sample was calculated by assuming collagen to contain 13.5% hydroxyproline by mass, and a molecular weight of 399 kDa (28, 29). When quantifying cross-links in bone, results are typically normalized to the collagen or hydroxyproline content of the sample, as collagen is the primary organic component of bone (3, 10, 11, 15, 17–19, 23, 30–36).

Statistics

Data were analyzed with JMP software (SAS, Cary, NC) using a two-way ANOVA model including time, radiation dose, and their interaction as variables. Statistical significance was assumed at p ≤ 0.05. Post-hoc pairwise comparisons were conducted using Tukey’s post-hoc test, which accounts for multiple sampling errors.

RESULTS

One week after the last dose fraction, specimens subjected to 4×5 Gy demonstrated a significant increase in the extent of collagen glycation (number of cross-links per unit of collagen). Specifically, the quantity of pyridinolines (p = 0.008), pentosidine (p = 0.013), and non-specific AGEs (p = 0.020 for ex/em = 370/440 and p = 0.0196 for ex/em = 335/400) accumulated by each molecule of collagen were elevated in irradiated samples compared to non-irradiated controls at one week (Figure 1A–C). There were no significant differences between treatment groups at any other time point. Pentosidine, pyridinolines, and non-specific AGEs were all significantly affected by time (p < 0.001), although there was no consistent positive or negative trend with increasing time. The interaction between radiation and time was not significant.

Figure 1.

Degree of collagen glycation following radiation (4×5 Gy) treatment (average ± SD, n = 4–6 per treatment per time point). Data are expressed as quantity of cross-links per picomole of collagen. At 1 week post-radiation, irradiated samples had significantly elevated content of (A) pentosidine, (B) pyridinolines, and (C) non-specific AGEs compared to controls. * denotes significantly different compared to non-irradiated controls at the same time point (p < 0.020). # denotes significantly different from all other time points (p < .013).

Radiation treatment did not significantly impact tibial collagen content (ug of collagen per mg of dry decalcified bone, p = 0.977, Figure 1D). Tibial collagen content was significantly affected by time (p < 0.001), with both control and irradiated groups demonstrating a significant increase in collagen content at two weeks compared to all other time points (p ≤ 0.013). Collagen content at one week was significantly higher than at four (p < 0.001) and eight weeks (p = 0.020).

DISCUSSION

Previously, we have documented radiation-induced alterations in bone quality and microarchitecture, including loss of trabecular bone, thickening of cortical bone, increased mineral density, and transient increase in osteoclast numbers followed by loss of viable osteoclasts (1, 37–41). The overall increase in bone volume and mineral density following radiotherapy is accompanied by a decrease in mechanical strength, indicating that radiation induces alterations to the material properties of the bone matrix (1). The cause of the post-radiation bone embrittlement is not yet fully understood, but there is evidence that alterations to the organic matrix may contribute significantly to altered mechanical properties.

There is extensive evidence that cross-linking and glycation-associated changes to collagen are related to the biomechanical properties of bone, particularly in the context of aging and diabetic pathologies. Garnero et al. induced collagen cross-linking in ex vivo bovine cortical femur specimens, and found that increased pyridinolines and pentosidine per unit of collagen correlated with reduced compressive yield stress and post-yield energy absorption; stiffness remained unaffected (3). Similarly, Tang et al. found that AGE accumulation in cadaveric trabecular bone specimens correlated with decreased stiffness of individual trabeculae and overall decreases in post-yield strain energy and energy dissipation (15). Stiffening of the collagenous matrix is strongly associated with accumulation of cross-links in bone, contributing to increased skeletal fragility and fracture risk (17, 34). Elevated pentosidine content in diabetic rats has been correlated with decreased modulus, energy absorption, and peak load in three-point bending (11). Viguet-Carrin et al. demonstrated no direct association between collagen cross-links and compressive mechanical properties (stiffness, failure load, work to fracture) for intact cadaveric vertebral bodies. Bivariate analysis of their data, however, indicated a significant association between increased failure load and high bone mineral density and low pentosidine content (35). There is also evidence that the type of cross-links accumulated in collagen may be important. Enzymatic and non-enzymatic cross-links may have differential effects in determining mechanical properties of bone (9, 20, 32, 42).

Reports of post-radiation changes to the organic matrix of bone suggest multiple factors may contribute to modification of collagen integrity. Specifically, it has been suggested that radiation induces increased collagen fragmentation, but not tissue collagen content. Açil et al. reported radiation-induced collagen fragmentation following irradiation of porcine mandibles ex vivo (21). Our data demonstrate no radiation-dependent changes in overall bone collagen content. This is consistent with the observations of Açil et al. in bone (murine model, 9.5 Gy focal hindlimb), Sassi et al. in skin (breast cancer patients, 30–56 Gy), and Lindburg et al. in cultured articular cartilage explants (porcine, 2 Gy) (43–45).

Documentation of radiation effects on collagen cross-linking and glycation in vivo is particularly sparse. Açil et al. treated rat hindlimbs with a single 9.5 Gy radiation exposure (BED = 42.8 Gy_2.8, equivalent to 12 fractions of 2 Gy each, as calculated by us) and evaluated hindlimbs at 14 and 100 days post-RTx for hydroxyproline, lysylpyridinoline, and hydroxylysylpyridinoline content. The only radiation-associated effect was a decrease in lysylpyridinoline quantity per mg of bone at 14 days post-RTx, which returned to control levels by 100 days post-RTx (43).

In this study, we demonstrate an early, transient increase in both enzymatic (pentosidine, non-specific AGEs) and non-enzymatic (pyridinolines) cross-linking of bone collagen following focal radiation therapy. These data are consistent with our Raman spectroscopic results using this mouse model of focal radiotherapy (4×5 Gy), showed significantly increased collagen crosslink ratios, significantly decreased matrix depolarization ratios, and early decreased mineral depolarization ratios in irradiated tibias beginning at one week post-RTx (46). The current results also provide a potential at least partial explanation for the findings of Wernle et al., who subjected irradiated mouse femurs (single fraction doses of 0, 5, and 20 Gy) to compressive mechanical testing (1). Failure load was significantly increased in irradiated samples at 1 week, falling below that of controls at 12 weeks. Temporally this correlates with our findings of increased collagen glycation at 1 week post-RTx and fits with the wider body of literature documenting the roles of collagen glycation and cross-linking in regulating bone mechanical properties. Although Wernle et al. documented radiation-associated morphological changes, finite element modeling indicated that morphology alone insufficiently explained the altered mechanical properties. Incorporation of an embrittled failure model improved the predictive strength of the computation models, suggesting that material properties play a significant role in determining the mechanical integrity of bone post-radiation (1).

We hypothesize that accumulation of advanced glycation end products in irradiated bone may contribute to embrittlement of the collagenous matrix and thereby increase fracture risk. Equilibration of glycation product levels beyond the one-week time point in this animal model is likely the result of increased osteoclastic bone resorption that follows radiation exposure (beginning 2 to 4 weeks post-RTx) and continued deposition of new mineral (41, 47). Longer term, radiation induced bone fragility may be regulated by the interaction of altered bone morphology, vascularity, biochemical matrix alterations, and cellular activity. Future investigations will pursue identification of specific biological mechanisms contributing to post-radiation bone embrittlement, including the role of reactive oxygen species, cell-mediated matrix remodeling, and potential therapeutic interventions.

LIST OF ABBREVIATIONS

AGEs

advanced glycation end products

AU

arbitrary fluorescence units

ANOVA

analysis of variance

BED

biologically equivalent dose (Gy)

ex/em

excitation/emission wavelength (nm)

RTx

radiation treatment