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Osteolytic and mixed cancer metastasis modulates collagen and mineral parameters
within rat vertebral bone matrix
Burke M., Atkins A., Akens M., Willett T., and Whyne C.
Version Post-Print/Accepted Manuscript
Citation (published version)
Burke M, Atkins A, Akens M, Willett T, Whyne C. Osteolytic and mixed cancer metastasis modulates collagen and mineral parameters within rat vertebral bone matrix. J Orthop Res. 2016 Mar 30. doi: 10.1002/jor.23248. [Epub ahead of print]. PMID: 27027407
Publisher’s Statement This is the peer reviewed version of the following article: Burke M, Atkins A, Akens M, Willett T, Whyne C. Osteolytic and mixed cancer metastasis modulates collagen and mineral parameters within rat vertebral bone matrix. J Orthop Res. 2016 Mar 30, which has been published in final form at https://dx.doi.org/10.1002/jor.23248. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
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Osteolytic and mixed cancer metastasis modulates collagen and
mineral parameters within rat vertebral bone matrix
Authors
Mikhail Burke1,2, Ayelet Atkins1, Margarete Akens3,4,Tom Willett2,3, Cari Whyne1,2,3
1Orthopaedics Biomechanics Laboratory, Sunnybrook Research Institute, Toronto, ON; 2Institute
of Biomaterials and Biomedical Engineering and 3Department of Surgery, University of Toronto,
Toronto, ON; 4Techna, University Health Network, Toronto, ON
Corresponding Author: Dr. Cari Whyne, Orthopaedics Biomechanics Laboratory, Sunnybrook
Research Institute, 2075 Bayview Ave., Room S620, Toronto, ON M4N 3M5. Phone: 416-480-
6100, ext. 5056. Email: [email protected].
Author Contribution Statements:
Mikhail Burke – Primary Author. Responsible for co-ordinating resources, study design, sample
preparation prior to analysis, data and statistical analysis and drafting the paper.
Ayelet Atkins – Responsible for Raman spectrographic study design and data acquisition and
review of paper.
Margarete Akens – Contributed to study design. Responsible for tumour cell line maintenance
and inoculation of rats with tumour cells to optimize vertebral metastases growth and review of
paper.
Tom Willett – Contributed to study design. Facilitated HPLC data acquisition and helped to
critically revise paper draft.
Cari Whyne – Senior Author. Developed study design. Provided direct links to study resources,
guidance on data analysis, critically revised paper draft.
All authors have read and approved the final submitted manuscript.
Abstract
Metastatic involvement in vertebral bone diminishes the mechanical integrity of the spine,
however, minimal data exists on the potential impact of metastases on the intrinsic material
characteristics of the bone matrix. Thirty-four (34) female athymic rats were inoculated with
HeLa (N=17) or Ace-1 (N=17) cancer cells lines producing osteolytic or mixed (osteolytic &
osteoblastic) metastases respectively. A maximum of 21 days was allowed between inoculation
and rat sacrifice for vertebrae extraction. High performance liquid chromatography (HPLC) was
utilized to determine modifications in collagen-I parameters such as proline hydroxylation and
the formation of specific enzymatic and non-enzymatic (pentosidine) cross-links. Raman
spectroscopy was used to determine relative changes in mineral crystallinity, mineral
carbonation, mineral:collagen matrix ratio, collagen quality ratio and proline hydroxylation.
HPLC results showed significant increase in the formation of pentosidine and decrease in the
formation of the enzymatic cross-link deoxy-pryridinoline within osteolytic bone compared to
mixed bone. Raman results showed decreased crystallinity, increased carbonation and collagen
quality(aka 1660/1690 sub-band) ratio with osteolytic bone compared to mixed bone and healthy
controls along with an observed increase in proline hydroxylation with metastatic involvement.
The mineral:matrix ratio decreased in both osteolytic and mixed bone compared to healthy
controls. Quantifying modifications within the intrinsic characteristics of bone tissue will provide
a foundation to assess the impact of current therapies on the material behaviour of bone tissue in
the metastatic spine and highlight targets for the development of new therapeutics and
approaches for treatment.
Introduction
Metastatic involvement in vertebral bone occurs in up to 1/3 of cancer patients1 and can manifest
as bone-resorbing (osteolytic), bone forming (osteoblastic) or a mixture of the two. Vertebral
metastasis has been linked with diminished mechanical integrity of the spine with bone
metastases leading to a high incidence (~66%) of skeletal related events (SREs), which can
significantly impact quality of life and physical function2.
Previous work has focused on structural, mechanical and mineral density changes in
metastatically involved bone with respect to the risk and pattern of fracture3. Significant co-
relations between specific CT-based stereological parameters and mechanical behaviour have
also been established4.However, these detected modifications in total bone structure or mass may
not fully account for observed SREs due to modified mechanical behaviour. The intrinsic
material characteristics of bone has been shown to be impacted by pathological conditions such
as type-I diabetes5 and should be accounted for in metastatic involvement.
Bone is a complex two-phase composite material whose material properties are dependent on
both its organic (collagen) and mineral (hydroxyapatite) components and their combined
ultrastructure. In its organic phase, the rectilinear array of collagen type-I fibrils provides bone
with enhanced ductility increasing the tissue's toughness6. This observed fibrillar structure, into
which the helical collagen-I molecules aggregate, is maintained and stabilized in part by the
formation of covalent cross-links between these molecules. These cross-links can be formed
through enzymatic pathways connecting the non-helical telopeptides of adjacent collagen
molecules7 to form immature divalent cross-links or mature trivalent cross-links or through non-
enzymatic pathways connecting the helical peptides8. A variety of enzymatic and non-enzymatic
cross-links exist within bone but studies tend to focus on the analysis of the trivalent mature
enzymatic pyridinium cross-links: pyridinoline (pyr) and deoxy-pyridinoline (dpyr); and the non-
enzymatic crosslink: pentosidine (pen)5, 9, 10. This focus is due to the natural fluorescence of these
cross-links making them easily identifiable markers in high performance liquid chromatography
(HPLC)5, 11. Decreases in pyridinium cross-links has been associated with a reduction in bending
strength and the elastic modulus of bone12. Modifications in the ratio between immature cross-
links and mature pyridinium cross-link levels within bone have been associated with changes in
observed bone strength or toughness9. Increase in non-enzymatic crosslinks have been shown to
negatively impact bone ductility, toughness and post-yield properties13-15. While no data exists
for metastatically involved bone, pathologic bone secondary to diabetes and osteoporosis has
demonstrated increases in pen levels suggesting pen level’s potential as a biomarker to
pathologic bone tissue.5, 9, 16. Note, these studies utilized bone samples for direct and absolute
measurement of cross-link concentration. While this allows for assessment of cross-link changes
due to both bone turnover and pathologically formed bone, pen levels can be inferred by
concentration detected in urine or serum due to turnover8.
Formation of these cross-links could be impacted by numerous factors. An imbalance between
produced reactive oxygen species (ROS) and the local system's ability to counteract with
sufficient anti-oxidising mechanisms and repair potential damage (known as oxidative stress)
could provide an environment conducive to oxidative reactions associated with advanced
glycation endproduct (AGE) formation9, 17. Additionally, hydroxylation of residues such as lysine
in the collagen molecule are critical in dictating the pathways of enzymatic cross-link
formation18. Modifications in collagen cross-links and hydroxylation of residues are detectible
via techniques such as high powered liquid chromatography.
Bone's mineral component primarily exists as hydroxyapatite crystals, which nucleate and grow
within the spacing between collagen fibrils. This mineral presence within bone is critical in
providing bone its strength and stiffness. While it has been theorized that mineral dimensions
would impact bone stiffness, studies have shown conflicting data between modified mineral size
and mechanical behaviour19. Modified chemistries of the calcium mineral can occur due to ionic
substitution of phosphate ion within the environment; the most common of those being with the
carbonate ion, which make up roughly 7wt% of the apatite lattice20. This type-B carbonate
substitution is suspected to be driven by the presence of non-collageneous proteins in bone and
the presence of carbonate ions has been linked to modifications of metabolic activity within
bone's localized biosystem such as buffering acidity potentially connected with bone resorption21
and stimulating bone growth22. Carbonate within the apatite structure has also been associated
with a modification in crystal structure with observed decreases in crystal dimensions or
increased lattice strain23. Some work has started to study the impact of metastasis on the mineral
crystal structure through Raman spectroscopy showing patterns of decreased crystallinity, crystal
size and increased carbonation24. This impact could also be cyclic in nature as factors such as
crystallinity and crystal size have been shown to impact metastatic cell behaviour25, 26. Raman
spectroscopy can also be utilized to assess the mineral/collagen ratio within bone tissue
indicative of tissue mineral density instead of observing total bone mass.
To understand the material behaviour of metastatically involved bone requires evaluation of
changes which occur in both organic and mineral phases. As such, this study aims to evaluate
collagen cross-linkage and mineral crystal structure in osteolytic and mixed metastatically
involved vertebral bone using well established in vivo rat models27, 28. It is hypothesized that due
to the associated increase of reactive oxygen species (ROS) production with cancer cells 29, the
increase in the oxidative stress within the bone microenvironment due to metastatic involvement
would lead to increase oxidation reactions and hence an increase in the formation of the non-
enzymatic pentosidene. Furthermore, an increase in the hydroxylation of specific collagen amino
residues is hypothesized to occur within metastatically involved bone as cancer cells have been
associated with an increase in the expression of hydroxylation enzymes such as and prolyl
hydroxylase (PH)30 and lysyl hydroxylase (LH)31. Hydroxylation impacts enzymatic cross-
linkage pathways as the cross-linkage formed differs by whether substrates/intermediates were
hydroxylated8. Additionally, previous work utilizing a murine metastatic lung-cancer model has
linked metastatic involvement to increased expression of LH2 and subsequent modification in
enzymatic collagen cross-link concentrations31. Factors such as hypoxia-inducible factor–1(HIF-
1) and signal transducer and activator of transcription 3 (STAT3), which were linked to the
observed increase in LH2, are also associated with the development or presence of bone
metastases32, 33. This, combined with the fact that the coexpression of LH and lysyl oxidase (LO),
an oxidizing enzyme key in the formation enzymatic crosslink intermediates, can occur because
both genes are targets for HIF-1 binding and are upregulated by extracellular signals associated
with metastatic behaviour31, and increases the likelihood of observed changes in the quantity of
various enzymatic cross-links formed. Finally, we predict decreased crystallinity, collagen
mineralization, crystal size and increased carbonation with metastatic involvement based on
trends observed previously24.
Material/Methods
Animal model and metastatic inoculation: Well established rat models for studying spinal
metastases were utilized27, 28, 34. These models use systemic inoculation of tumour cells to
simulate the physiologic development of vertebral metastases. The animal use protocol was
approved by the Ontario Cancer Institute prior to performing the study. Randomly assigned 5–6
week old athymic female rnu/rnu rats were inoculated with human HeLa cervical cancer cells
(previously misidentified as MT-1 cells) to create osteolytic metastases or canine Ace-1 prostate
cancer cells to produce mixed (osteolytic/osteoblastic) metastases (N=17 per group). An
additional 12 rats were used as healthy controls. The HeLa and Ace-1 cell lines were cultured in
RPMI and DMEM/F-12 media respectively at 37°C with 5% CO2 and stably transfected with the
luciferase gene to enable bioluminescent image monitoring of tumor growth within the rat via
subcutaneous injection of luciferin. Cultured cell viability was determined utilizing a trypan blue
exclusion. Rats were anesthetised using nose-cone inhalation of a 2% halothane/air mixture.
Intracardiac injections containing ~1.5×106 cells (in 0.2mL of media) were injected into the left
heart ventricle. After 14 and 21 days, bioluminescence imaging (Xenogen) was performed for
detection and semi-qualitative assessment of the degree of metastatic growth within the rat, and
particularly in the spine. The rats were euthanized 21 days after initial inoculation via CO2
asphyxiation, their vertebrae harvested and wrapped in saline dampened gauze and stored in -
20˚C freezer until testing. In cases of premature rat death the vertebrae were removed
immediately and noted accordingly.
High Performance Liquid Chromatography: HPLC was performed to quantify crosslinks using
a previously published protocol11, 35, 36. Briefly, soft tissue was removed from extracted T12-T13
vertebrae via papain digestion. Samples were defatted and then hydrolyzed in 11 M HCl at
110°C for 24 h. Samples were diluted and added to a sample buffer containing 10% acetonitrile,
1% HFBA and water plus an internal standard (pyridoxine). Pyridinoline, deoxy-pyridinoline
and pentosidine were quantified against standards of pentosidine (PolyPeptide Group,
Strasbourg, France) and pyridinoline (Qiagen, Hilden, Germany). Agilent Zorbax Eclipse XDB-
C18 Reversed-Phase C18 HPLC columns were used (150 × 4.6 mm, 5 μm particle size, 80 Å
pore size, end-capped; Agilent Technologies, Mississauga, Canada). The mobile phase for
crosslink quantification incorporated A: 26% methanol+0.1% heptafluorobutyric acid (HFBA),
B: 85% acetonitrile+0.1% HFBA and C: 38% methanol +0.08% (HFBA) at different elution
times (A: 0-18 min, 40-50 min, B: 18-30min, C: 30-40 min). A separate HPLC method utilizing
the same type of column was used to measure collagen content via hydroxyproline quantification
of the same samples using both hydroxyproline and amino acid standards (Sigma-Aldrich).
Samples were diluted with borate buffer and homoarginine (internal standard) and underwent
derivatization via the cycled addition of fluorenylmethyloxycarbonyl chloride (FMOC-Cl) +
acetone, pentane and extraction of waste top layer. .For each sample run, an elution profile
measuring fluorescence vs. elution time was obtained. The areas under the peaks were measured
and compared to a standard curve in order to calculate the concentration of each crosslink,
hydroxyproline and proline. Cross-link concentration was then normalized to collagen content.
The degree of proline hydroxylation was also determined by hydroxyproline/proline ratio.
Raman Spectroscopy: Extracted T8 vertebrae were cut in the sagittal plane with a diamond blade
(Buehler, Illinois, USA) then polished with grit papers (600, 1200, 2400, 4000 and diamond
paste 3um and 1um). The bone specimens were thawed in a 4°C refrigerator 24 hours prior to
Raman spectra acquisition. Acquisition was done with an inVia Confocal Raman Microscope
(Renishaw, Gloucestershire, UK) which includes a Renishaw spectrometer coupled into a
DMI6000 epifluorescence microscope (Leica, Wetzar, Germany). A 785 nm laser light was used
to focus on the vertebral body through a Leica HC PLAN APO 20x/0.70NA (laser power 3 mW,
spot size 4um in the y direction and 0.5um in the x direction). The bone sample was kept wet
with PBS, each sample had 3-5 locations tested and 4-6 spectra were acquired from each location
(Figure 1a), consisting of two accumulations with an exposure time of 15s. The number of
testing points chosen reflected that which was utilized in previous studies24 and point locations
were chosen on trabecular bone adjacent to abnormal voids in trabecular structure attributed to
metastatic involvement. Custom script written in Matlab was used to correct the baseline of each
spectrum. Peak intensities of phosphate ν1 (959cm−1), carbonate (1070cm−1), proline (855cm−1),
hydroxyproline (876cm−1) and amide-1(1665-1670cm−1), were determined for the mineral and
collagen properties of the bone (Figure 1b)24. For peak analysis, background signal noise was
removed by subtracting a baseline polynomial curve that best fit the data. To verify the results, a
second derivative of the spectrum was taken to attenuate the low-frequency components. Three
sub-bands (1650-1660cm−1, 1665-1670cm−1, 1680-1690cm−1) in the amide-1 region (1650-
1690cm-1) were identified and were Gaussian in nature.
From these intensities, a number of ratios were calculated for analysis. The mineral/matrix ratio
(phosphate ν1/proline+hydroxyproline) and carbonate/matrix ratio (carbonate/
proline+hydroxyproline) illuminate the degree of mineralization of the bone tissue.
Proline+hydroxyproline were chosen as the matrix factors to utilize in the mineral/matrix and
carbonate/matrix ratios due to those factors having a distinct tie to collagen-I37. Mineral
crystallinity (1/FWHM of phosphate ν1 peak), carbonate/phosphate ν1 ratio (indicative of
mineral carbonation), hydroxyproline/proline ratio (indicative of proline hydroxylation), and the
collagen quality parameter (mature/immature enzymatic cross-link ratio) were also calculated
and compared between sample groups.
Statistical data analysis: For each data set, the normality of the data was evaluated utilizing the
Shapiro-Wilk test. If the normality assumption held, then a test for the homogeneity of variance
between groups via Levene's test was performed. In data sets without equal variance, Welsh
followed by Tamhane post hoc tests for multiple comparisons were performed to test for
differences between sample groups. When equal variance and normality could be assumed,
analysis of variance followed by the Tukey post hoc test for multiple comparisons between
samples groups were used. In cases where the normality assumption could not be held, the
Kruskal-Wallis H test followed by the Dunn-Bonferroni post-hoc approach for multiple
comparisons between sample groups were used. All data are presented as mean±standard
deviation, with a significance level of p<0.05 (SPSS v23, SPSS, Chicago, IL, USA).
Results
Metastatic Involvement: Bioluminescence analysis found that 14/17 rats injected with HeLa
cells (~82% success rate) and 12/17 rats injected with Ace-1 cells (~71% success rate) displayed
metastatic tumour growth in the spine (including the approximate location of the T8-T13
vertebrae as displayed in Figure 2), as well as in the heart, lungs and femurs. The degree of
metastatic growth in each rat was random. A total of 25 rats (HeLa: N=9; Ace-1: N=7; Healthy:
N=9) were utilized for HPLC analysis. For Raman, 25 samples were used (HeLa: N=10; Ace-1:
N=9; Healthy: N=6).
Cross-link and Proline Hydroxylation Quantification via HPLC: The non-enzymatic cross-link
pentosidine showed a large statistically significant increase (2.4x, p<0.05) in observed pen levels
in osteolytic vertebrae compared to healthy controls (Figure 3a). Although total pyridinium
cross-links content showed no significant difference between healthy and metastatic involved
bone, a trend (p = 0.087) was observed between osteolytic and mixed bone samples (Figure 3b).
Deoxy-pyridinoline levels were significantly lower (p<0.05) in the osteolytic compared to the
mixed vertebrae (Figure 3c). There were no significant differences in pyridinoline levels between
the 3 groups (Figure 3d). There was a significant increase in hydroxyproline formation (~16%,
p<0.05) in osteolytic bone compared to healthy controls (Figure 4a) with no statistically
significant changes in proline concentration (Figure 4b). Osteolytic bone samples demonstrated a
trend toward an increase (~6%) in the hydroxyproline/proline ratio (Figure 4c) compared to
mixed bone and healthy controls (p = 0.053, p = 0.077, respectively).
Modifications in Mineralization, Collagen Quality and Proline Hydroxylation Ratios assessed
via Raman: Modifications in both crystallinity and crystal structure were found due to metastatic
involvement. The 1/FWHM value of ν1Phosphate peak showed a small but statistically
significant decrease (~3%) in the osteolytic group compared to both the mixed model and
healthy controls, indicative of decreased crystallinity (Figure 5a). The carbonate/phosphate ratio
in the osteolytic samples was elevated (~6%) compared to the other groups, implying increased
carbonate ion substitution in the crystal lattice (Figure 5b).
The mineral/matrix ratio showed a statistically significant decrease in the presence of the
phosphate component of hydroxyapatite relative to the proline/hydroxyproline residues specific
to collagen-I for both osteolytic (~ 15%) and mixed groups (~11%) versus healthy controls
(Figure 5c). There was also a decrease in the carbonate/matrix ratio in bone with metastatic
involvement (Figure 5d).
The collagen quality parameter, thought to be indicative of the level of the ratio between mature
and immature cross-links, was moderately reduced (5%) in osteolytic samples compared to both
mixed and healthy controls (Figure 5e). Additionally, there were observed differences in the
hydroxyproline/proline ratio between all three samples groups with osteolytic samples exhibiting
the highest ratio (~ 10% increase) (Figure 5f).
Discussion
Both the organic and mineral phases of metastatically involved vertebrae demonstrated changes
as compared to healthy controls, suggesting that tumour induced changes impact the remaining
bone at a material level. The most striking differences were seen in pentosidine (pen) levels. Pen
is an AGE crosslink between lysine and arginine found in the helical domains of adjacent
collagen molecules. Pathological conditions such as Type 1 and Type 2 diabetes considered to
negatively impact bone quality have been shown to modify the quantity of collagen cross-link
formed within bone tissue5, 9. In each case, pen levels were shown to increase. This, combined
with increased pen levels being associated with diminished bone mechanical properties13, 14,
implies an importance of pen as a potential biomarker for poor bone quality and a risk factor for
fracture. In our study, osteolytic metastatic involvement within the vertebrae (HeLa) was shown
to greatly increase the formation of pen in the bone. In general, an increase in ROS species
concentration associated with the presence of cancer cells29could facilitate the oxidation of open
chain aldehyde metabolic intermediates leading to the production of AGEs such as pen.
However, Ace-1 metastatic involvement did not impact pen levels in a statistically significant
manner. This contrast to the HeLa involvement could be due to variability in tumour burden
among samples. An increase in pen has been linked to diminished bone quality and may act as a
marker for oxidative stress and AGE production which can impact bone remodelling13, 14.
Amplified concentrations of ROS can damage the extracellular matrix (collagen) and impact
cellular metabolic reactions (osteoblasts and osteoclasts remediated bone remodelling)38.
Collagen degradation, both in terms of fragmentation and susceptibility to enzymatic
degradation, has been shown to be facilitated by ROS presence39, 40. All these factors combined
with other AGE presence may have an impact on bone's mechanical behaviour.
Pyridinoline (pyr) and deoxypyridinoline (dpyr) are formed via a hydroxyallysine dominant
(ahyl) pathway. Since ahyl versus allysine (alys) formation is dependent upon the availability of
hydroxylysine (hyl), both the action of lysyl hydroxylases (LH) and lysyl oxidases (LO) on
lysine (lys) are important in the formation of these cross-links. The level of Lys hydroxylation
has been shown to play an important role in the tissue-specific pattern of enzymatic cross-links
and defining the functionality of type I collagen in tissue18. Metastatic involvement in other
systems has been linked to increased LH and LO levels within the microenvironment31.
Additionally, ROS presence could also induce non-enzymatic hydroxylation of amino residues41.
Therefore, one might anticipate an increase in the formation of ahyl dominant enzymatic cross-
links with metastatic involvement. However, this was not the case for the observed mature
enzymatic cross-links with no significant difference between osteolytic or mixed bone compared
to healthy bone. The significant relative decrease in dpyr formation in osteolytic compared to
mixed bone however, does suggest that collagen cross-link formation pathways are impacted
differently by the type of cancer involved. Although such modifications in enzymatic cross-
linking (both mature and immature) have been shown to impact bone mechanical behaviour 12, 42,
the presence or directionality of the change in pyridinium cross-link levels in bone can differ
under different pathological conditions5, 9, 10, 43. Note, post-hoc power analysis showed that the
comparison of dpyr differences was underpowered (power=0.48). This may have impacted the
ability to detect a statistically significant difference between osteolytic and healthy bone
(p=0.114). This general decrease in pyridinium cross-link formation is likely due to competitive
utilization of lys and hlys residues in AGE formation creating a deficiency in the formation of
enzymatic cross-links. The emphasis on dpyr decrease compared to pyr might be due to the
potential increase in the hydroxylation of lysine, limiting the available of helical lys which is
required in dpyr formation but not for pyr formation. Unfortunately, we did not measure lysine
hydroxylation in this study.
Hydroxylation of proline in collagen is mediated by the enzyme prolyl hydroxylase. The
presence of hydroxyproline in collagen is critical for the stability of the collagen triple helix via
hydrogen bonding interactions. Additionally, the polar interactions between protruding hydroxyl
groups of collagen molecules within collagen fibrils could impact both the chemical and
observed physiological properties of the fibril. For example, it has been shown that
hydroxylation is favourable for HA nucleation and growth44. The expression of prolyl
hydroxylase has been linked to cancer metastases in primary tumours often associated with bone
metastasis, likely to facilitate increased extracellular matrix production to providing structure for
tumour cell adhesion, growth and taxis30, 45. Additionally, ROS species associated with metastatic
involvement could potentially lead to non-enzymatic hydroxylation of amino residues41.
Therefore metastases could facilitate an increase in non-enzymatic hydroxylation and the prolyl
hydroxylase levels in the bone micro-environment promoting increased hydroxyproline
expression and the observed increase in the hydroxyproline/proline ratio in metastatic bone
groups compared to healthy controls. Note that in this study, the modifications in
hydroxyproline/proline ratio observed among sample groups was different between HPLC and
Raman. HPLC provides absolute values on the quantity of proline and hydroxyproline residue
within all bone in the vertebrae whereas Raman analysis does not provide absolute values (just
the ratio), is focal in nature, and locations of analysis were chosen by their likely proximity to
tumour. This difference in data collection methodology could account for Raman’s perceived
increased sensitivity in detecting differences in the hydroproline/proline ratio in metastatically
involved bone and suggests that tumour impact on bone collagen and bone is proximal in nature.
Note, although hydroxyproline levels may be modified with metastatic involvement, its
expression as the gold standard in measuring collagen content was utilized.
Proline and hydroxyproline were chosen as the matrix factors to utilize in the mineral/matrix and
carbonate/matrix ratios in Raman due to those factors having a distinct tie to collagen-I37. The
observed decrease in the mineral/matrix ratio for both the osteolytic and mixed bone samples
agrees with decreases in tissue mineral density associated with metastatic involvement observed
via other modalities (i.e. µCT scanning + segmentation , pycnometry + mass measurements)4, 27,
34.
Metastatic involvement has been previously shown to both influence and be influenced by
hydroxyapatite (HA) crystallinty and crystal size25, 26. Our results revealed a decrease in
crystallinity in osteolytic samples compared to healthy controls (p<0.05) with a similar pattern
(p<0.05) in the mixed metastasis groups. This could be attributed to the observed increase in
carbonate/phosphate ratio, implying increased carbonate substitution crystal defects or may be
associated with changes in HA crystal dimensions. The observed increase in the relative
carbonation of the HA mineral could be indicative of the increased acidity of the local bone
biosystem due to both increased overall osteolytic activity associated with tumour involvement
in both metastatic models and the acidic extra-cellular fluid of the tumour tissue46. It has been
shown that acidic microenvironments favor the dissolution of phosphate compared to carbonate
in the cross-sectional areas of bone47.
In Raman spectroscopic analysis, the 1660cm-1 and the 1690 cm-1 peaks within the amide-1
region (1650-1690cm-1) are associated with the presence of mature pyr and immature deH-
DHLNL respectively. Although there is contention on the direct link of the 1660/1690 to the
ratio of mature/immature crosslinks in the bone48, it is still commonly used in Raman to provide
information on bone age and the quality of the collagen (as immature cross-links mature over
time). HPLC analysis showed no significant changes in pyr formation between the groups.
However, a decreasing 1660/1690 ratio in the osteolytic samples implies that there is a change in
the amount of immature cross-links formed in the collagen. This potential modification in the
ratio between mature and immature cross-links could impact bone’s material behaviour9.
While the results of this study show clear changes in the formation of collagen cross-links and
modifications in mineralization of the metastatically involved bone matrix, some limitations
must be noted with respect to the approach utilized. Although the presence of tumour within the
spinal area of inoculated rats was established via bioluminescence imaging, tumour burden was
not volumetrically quantified within each tested vertebrae. Therefore the potential impact of the
quantity of tumour on collagen-cross-link formation and mineralization was not evaluated. As
well, the tumour cell lines utilized were from different animal and tissue sources, however, the
immune compromised nature of the rats should minimize any impact of cell species differences
in the study. Additionally, potential sub differences between existing bone/new matrix and
osteolytic /osteoblastic bone tissue within the Ace-1 mixed metastatic model were not evaluated.
Raman provided qualitative, comparative assessment of bone quality between samples groups,
yet it does not provide absolute value assessment for specific phase characteristics (such as the
dimensional changes in the HA crystals). Finally, while pen is an AGE product and a
relationship between pen fluorescence and bulk fluorescence of all AGEs has been shown in
other studies, no clear relationship between pen formation and the formation of other non-
enzymatic cross-links has been proven
Conclusion
This study demonstrates that metastatic involvement has a clear impact on the formation of
specific non-enzymatic and mature enzymatic cross-links in vertebral bone. Future work will
need to link these cross-link modifications to potential changes in collagen microstructure, bone
mineralization and the overall material behaviour of the pathologic bone matrix. Additionally,
specific elucidation of potential differences between osteolytic and osteoblastic tissue will be
important in characterising focal impact and distinguishing sub-differences within mixed
metastatic involvement. Also, studying the relative impact of these tissue level changes to the
mechanical behaviour of vertebral bone is important for future analysis. Ultimately, by
quantifying the influence of cancer on bone tissue, a fundamental basis for understanding the
mechanics of structural changes can be established; which will be key in developing new
integrative strategies to mechanically model metastatically involved vertebral bone and highlight
areas of focus in the evaluation of new and existing treatment options.
Acknowledgements
Grant funding for this study was provided by the Canadian Institutes of Health Research (#68911
and #125886). The authors declare that there are no conflicts of interest.
Figure Legend
Figure 1 - a) Photograph of cross section of representative rnu/rnu rat T8 vertebrae. White
arrows point to Raman spectra collection points. Points were chosen on trabeculae adjacent to
seemingly larger abnormal voids in trabecular structure. b) Example of Raman spectrograph
from each sample group (osteolytic, mixed and healthy bone). Selective bands were marked with
biochemical assignments.
Figure 2 – Representative bioluminescent images of rats inoculated with HeLa (a) and Ace-1 (b)
21 days post-inoculation. Metastatic growth was observed in various regions of the rat anatomy
with the region of interest being the spinal column area (indicated by oval overlay). The dotted
square overlay indicates the approximate location of T8-T13 vertebrae.
Figure 3 - Quantitative assessment of a) pentosidine b) total pyridinium c) deoxypyridinoline
and d) pyridinoline collagen crosslinks concentrations from HPLC analysis of rat vertebral bone
within each sample group. Osteolytic bone showed increases in expression of the non-enzymatic
cross-link pentosidene and decreases in the mature enzymatic cross-link deoxypyridionline
compared to healthy and mixed bone respectively. A total of N=25 rats (HeLa: N=9; Ace-1:
N=7; Healthy: N=9) were assessed.
Figure 4 - Quantitative assessment of a) hydroxyproline and b) proline collagen residue
concentrations from HPLC analysis and the calculated c) hydroxyproline/proline ratio of rat
vertebral bone within each sample group. Osteolytic bone showed a statistical trend towards
higher hydroproline/proline ratios compared to healthy and mixed bone. A total of N=25 rats
(HeLa: N=9; Ace-1: N=7; Healthy: N=9) were assessed.
Figure 5 - Raman spectrograph-derived parameters of bone composition between sample groups.
Characteristics analyzed included a) mineral crystallinity, b) carbonate/phosphate, c)
mineral/matrix, d) carbonate/matrix ratios, e) the collagen quality parameter and f)
hydroxyproline/proline ratio. All parameters showed statistical difference between healthy and
metastatic groups. A total of N=25 rats (HeLa: N=10; Ace-1: N=9; Healthy: N=6) were assessed.
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