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RESEARCH ARTICLE
β-Aminopropionitrile-Induced Reduction in
Enzymatic Crosslinking Causes In Vitro
Changes in Collagen Morphology and
Molecular Composition
Silvia P. Canelo
´n
1
, Joseph M. Wallace
1,2,3¤
*
1Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, United States of
America, 2Department of Biomedical Engineering, Indiana University-Purdue University at Indianapolis,
Indianapolis, Indiana, United States of America, 3Department of Orthopaedic Surgery, Indiana University
School of Medicine, Indianapolis, Indiana, United States of America
¤Current address: Department of Biomedical Engineering, Indiana University-Purdue University at
Indianapolis, Indianapolis, Indiana, United States of America
*jmwalla@iupui.edu
Abstract
Type I collagen morphology can be characterized using fibril D-spacing, a metric which
describes the periodicity of repeating bands of gap and overlap regions of collagen
molecules arranged into collagen fibrils. This fibrillar structure is stabilized by enzymatic
crosslinks initiated by lysyl oxidase (LOX), a step which can be disrupted using β-aminopro-
pionitrile (BAPN). Murine in vivo studies have confirmed effects of BAPN on collagen nano-
structure and the objective of this study was to evaluate the mechanism of these effects in
vitro by measuring D-spacing, evaluating the ratio of mature to immature crosslinks, and
quantifying gene expression of type I collagen and LOX. Osteoblasts were cultured in com-
plete media, and differentiated using ascorbic acid, in the presence or absence of 0.25mM
BAPN-fumarate. The matrix produced was imaged using atomic force microscopy (AFM)
and 2D Fast Fourier transforms were performed to extract D-spacing from individual fibrils.
The experiment was repeated for quantitative reverse transcription polymerase chain reac-
tion (qRT-PCR) and Fourier Transform infrared spectroscopy (FTIR) analyses. The D-spac-
ing distribution of collagen produced in the presence of BAPN was shifted toward higher D-
spacing values, indicating BAPN affects the morphology of collagen produced in vitro, sup-
porting aforementioned in vivo experiments. In contrast, no difference in gene expression
was found for any target gene, suggesting LOX inhibition does not upregulate the LOX gene
to compensate for the reduction in aldehyde formation, or regulate expression of genes
encoding type I collagen. Finally, the mature to immature crosslink ratio decreased with
BAPN treatment and was linked to a reduction in peak percent area of mature crosslink
hydroxylysylpyridinoline (HP). In conclusion, in vitro treatment of osteoblasts with low levels
of BAPN did not induce changes in genes encoding LOX or type I collagen, but led to an
increase in collagen D-spacing as well as a decrease in mature crosslinks.
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 1 / 13
a11111
OPEN ACCESS
Citation: Canelo
´n SP, Wallace JM (2016) β-
Aminopropionitrile-Induced Reduction in
Enzymatic Crosslinking Causes In Vitro Changes in
Collagen Morphology and Molecular Composition.
PLoS ONE 11(11): e0166392. doi:10.1371/journal.
pone.0166392
Editor: Laurent Kreplak, Dalhousie University,
CANADA
Received: August 4, 2016
Accepted: October 27, 2016
Published: November 9, 2016
Copyright: ©2016 Canelo
´n, Wallace. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by funding
from the NIH (1K25AR067221-01A1), IUPUI
departmental start-up funds, Research Support
Funds Grant from the IUPUI Office of the Vice
Chancellor for Research and funding from the
IUPUI Biomechanics and Biomaterials Research
Center.
Competing Interests: The authors have declared
that no competing interests exist.
Introduction
Bone is a composite material made up of an inorganic (hydroxyapatite mineral) phase, a pro-
teinaceous organic phase, and water. Comprising 90% of bone’s organic phase, type I collagen is
the most abundant protein in the human body [1]. Both hydroxyapatite and collagen contribute
to bone mechanical properties; hydroxyapatite provides compressive strength and stiffness
while collagen provides tensile strength and ductility [2–4]. Because bone is a hierarchical mate-
rial, changes in the properties of either phase can influence bulk mechanical properties of the tis-
sue and bone structure. In some cases, these effects can compromise bone’s ability to serve its
structural function of bearing dynamic loads associated with movement. For example, decreased
bone strength is a characteristic of osteoporosis and reflects deterioration in bone density and
bone quality [5–7]. Osteogenesis imperfecta is also characterized by decreased bone strength
and toughness, and is caused by disruptions in the quality or amount of type I collagen [8–10].
Type I collagen in bone is synthesized by mature osteoblasts as a right-handed helical struc-
ture formed from three polypeptide chains of amino acids. Each chain is a left-handed helix
with repeating Gly-X-Y triplets where Gly is glycine, X is usually proline, and Y hydroxypro-
line [11,12]. In type I collagen molecules, two of these polypeptide chains are α1 helices and
one is an α2 helix. Once a triple-helical molecule forms, N and C terminal ends are cleaved by
proteinases, leaving mature collagen molecules. These molecules self-assemble in line with one
another into microfibrils, then in parallel into quarter-staggered arrays with overlap and gap
regions, and finally into three-dimensional fibrils. The overlap and gap regions produce an
oscillating surface topography of axially repeating bands along the fibril length, referred to as
the D-spacing or periodicity of the fibril [13] (Fig 1). This D-spacing is a morphometric char-
acteristic of collagen fibrils and exists as a distribution of values near the theoretical 67 nm[14].
Changes in mean D-spacing or its distribution of values can be used to detect differences in
collagen structure, tissue origin, and hydration state [15–19].
Post-translationally, collagen fibrils are stabilized within their staggered array by intramo-
lecular and intermolecular crosslinks [20–22]. Enzymatic crosslink formation begins when tel-
opeptide lysine and hydroxylysine precursors, through lysyl oxidase (LOX) initiation, convert
to telopeptide aldehydes, allysine and hydroxyallysine, respectively [21,23]. The allysines, in
combination with other precursors (i.e. helical lysine or hydroxylysine) form covalent chemi-
cal crosslinks. Some of these crosslinks mature to their trivalent form as pyridinolines and pyr-
roles. Crosslink synthesis can be limited by compounds such as penicillamine and β-
aminopropionitrile (BAPN), resulting in a crosslink deficiency which characterizes a disease
known as lathyrism [24–26]. BAPN is found in the seeds of the lathyrus odoratus plant, grown
as a famine crop, and acts by irreversibly binding to the LOX active site. This binding prevents
LOX from catalyzing aldehyde formation and subsequently blocks the formation of new cross-
links and the maturation of pre-existing immature crosslinks.
While BAPN has been shown to affect macroscale bone properties and nanoscale properties
of type I collagen in vivo in rabbit, rat, and mouse models [27–29], knowledge of its direct
effects on the morphology, expression, and crosslinking of collagen produced in vitro by osteo-
blasts is limited [30,31]. Evidence in the literature shows LOX mRNA expression increases
with low levels of BAPN exposure, and decreases at higher concentrations as the differentia-
tion process is impaired [31–33]. At low concentrations, as bioavailability of LOX is decreased,
the cells may possess the ability to upregulate LOX expression to bring the level of available
LOX back within a normal range. Few studies have investigated BAPN inhibition of LOX to
block crosslink formation in vitro [30–32], and fewer still specifically attribute the effect to a
change in immature or mature crosslinks [31]. The purpose of this study was to modify a key
step in post-translational collagen synthesis to observe alteration to type I collagen in its native
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 2 / 13
state. It was hypothesized that BAPN-induced inhibition of collagen crosslinking would (1)
alter the D-spacing morphology of collagen produced in vitro by osteoblasts (2) drive upregu-
lation of the LOX gene to compensate for the reduction in aldehyde formation, leading to an
observed increase in mRNA expression and (3) inhibit the formation of mature crosslinks,
specifically hydroxylysylpyridinoline.
Materials and Methods
Cell culture
MC3T3-E1 Subclone 4 (ATCC CRL-2593) murine preosteoblasts were obtained from the
American Type Culture Collection (ATCC, Manassas, VA) and cultured in proliferation
medium composed of αminimal essential medium (α-MEM, Life Technologies, Carlsbad,
CA), 10% fetal bovine serum (FBS, GIBCO, Carlsbad, CA), 0.5% penicillin/streptomycin
(GIBCO, Carlsbad, CA), and 1% L-glutamine (Hyclone, Logan, UT). MC3T3-E1 differentia-
tion control medium consisted of proliferation medium supplemented with 50 μg/ml ascorbic
acid (Thermo Fisher Scientific,Waltham, MA) and differentiation experimental medium was
additionally supplemented with 0.25 mM BAPN-fumarate (Sigma Aldrich, St. Louis, MO) for
crosslink inhibition experiments.
Fig 1. Collagen structure and organization. Collagen molecules self-assemble in a quarter-staggered array into microfibrils to form collagen
fibrils with characteristic periodic D-spacing.
doi:10.1371/journal.pone.0166392.g001
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 3 / 13
Collagen synthesis for analysis of collagen morphology
MC3T3-E1 cells were cultured in T75 flasks and allowed to proliferate for 3 days until reaching
80% confluence. Once confluent, the cells were seeded into 60 mm dishes at a density of
500,000 cells per dish. Cells were seeded into eight dishes, four of them control dishes without
BAPN and four treatment dishes with 0.25mM BAPN-fumarate. After seeding, the medium
was replaced and supplemented with ascorbic acid for 14 days to allow differentiation and pro-
mote collagen synthesis.
Atomic force microscopy (AFM)
Following 14 days of differentiation, the cultures were rinsed with phosphate-buffered saline
(PBS) and treated with 10 mM ethylenediaminetetraacetic acid (EDTA, Life Technologies,
Carlsbad, CA). Experiments in bone and tendon have shown that EDTA has negligible effects
on collagen fibril morphology (data not shown) and was here used to encourage cellular
detachment from the extracellular matrix (ECM) in order to expose the newly synthesized col-
lagen matrix for AFM imaging. After treatment with EDTA, the matrix was rinsed with ultra-
purified water (Milli-Q, EMD Millipore, Darmstadt, Germany) and allowed to dry. Five
locations within each dish were imaged with a Bioscope Catalyst Atomic Force Microscope
(Bruker, Santa Barbara, CA) in peak force tapping mode using ScanAsyst Fluid+ probes (Bru-
ker, Santa Barbara, CA). Within each location, a 30 μm x 30 μm scan was performed to find
areas where collagen was exposed by treatment with EDTA. A 10 μm x 10 μm scan was then
performed to find suitable areas for closer examination followed by a final 3.5 μm x 3.5 μm
scan of collagen fibrils appropriate for analysis. A 2 Dimensional Fast Fourier Transform
(2D-FFT) was performed to extract D-spacing from approximately 10 individual collagen
fibrils at each location, as previously described [16,34]. D-spacing analysis was performed on a
minimum of 50 fibrils per dish and 200 fibrils per experimental group.
Quantitative Reverse Transcription Polymerase Chain Reaction
An additional set of experiments was run for gene expression analysis by qRT-PCR. Cells from
a single flask were seeded into ten dishes (five control and five supplemented with 0.25mM
BAPN-fumarate). At the end of a 7 day differentiation period, the medium was removed from
each culture dish and replaced with 1mL of TRIzol reagent (Invitrogen, CA). RNA isolation
was performed using TRIzol reagent and reverse transcription (RT) was carried out using a
High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA). PCR was
performed using an ABI 7500 Fast PCR machine under the 9600 Emulation thermal cycling
mode with SYBR Green primers and Power SYBR Green PCR master mix (Life Technologies,
Carlsbad, CA). Primers were chosen for target genes encoding type I collagen α1 (COL1A1),
type I collagen α2 (COL1A2) and LOX as well as reference gene β-actin (BACT) [35,36]. Each
sample/gene combination was run in triplicate and water was used as the no-template control.
mRNA expression levels of the triplicates were averaged. Following an efficiency-calibrated
mathematical model [37], mRNA expression levels for each sample/target gene were averaged
and compared to the control group using the REST program [38]. The program calculates rela-
tive expression ratios using Eq 1 and employs randomization tests to obtain a level of signifi-
cance.
Ratio ¼ðEtargetÞDCTtargetðcontrolsampleÞ
ðEref ÞDCTref ðcontrolsampleÞð1Þ
E
target
and E
ref
are the qRT-PCR efciencies of a target gene and reference gene transcript,
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 4 / 13
respectively; and ΔCT is the difference in control and sample cycle thresholds for the respec-
tive gene transcript.
Fourier Transform Infrared Spectroscopy (FTIR)
Following the experimental methods described above, a third set of experiments was run to
analyze collagen’s secondary structure using FTIR. Cells were seeded into twelve dishes (six
control and six supplemented with 0.26mM BAPN). After 14 days of differentiation the dishes
were rinsed three times with PBS and three times with water. Samples were then directly trans-
ferred onto barium fluoride windows and air-dried.
FTIR spectroscopic analysis was performed using a Nicolet iS10 spectrometer (Thermo
Fisher Scientific, Waltham, MA). A water vapor background was collected and subtracted
from sample data as they were collected. Data were collected from the samples under nitrogen
purge at a spectral resolution of 4 cm
-1
. A minimum of three spectra were collected per sample
and they were averaged and treated as technical replicates. The amide I and amide II regions
(~1400–1800 cm
-1
) were baseline corrected according to published standards [39,40] using
OriginPro (OriginLab, Northampton, MA). Underlying peaks within these regions were
resolved as Gaussian peaks using second derivative analysis and each spectrum was curvefit
using GRAMS/AI (Thermo Fisher Scientific, Waltham, MA). The results from the converged
peak fitting were expressed as peak position and percentage area of the peak relative to the area
underneath the fitted curve. The investigation focused on peaks corresponding to positions at
~1660cm
-1
and ~1690cm
-1
, shown to be correlated to mature (HP, hydroxylysylpyridinoline)
and immature crosslinks, respectively [31,32,41,42].
Statistical analysis
All statistical analyses were performed using Statistical Analysis System (SAS Institute, Cary,
NC) and a value of p<0.05 was considered significant for all experiments.
To investigate difference in collagen fibril morphology due to the presence of BAPN, D-
spacing values measured from each culture dish were averaged to yield a single value. These
mean D-spacing values from control (n = 4) and treated (n = 4) samples were then compared
using a Mann Whitney Utest. This nonparametric test was chosen due to the low sample size.
To explore differences in the distribution of D-spacing values, the histogram and cumulative
distribution function (CDF) of each group was generated. The distributions of the control
(n = 217) and treated groups (n = 251) were compared using a k-sample Anderson-Darling
(A-D) test as previously described [43].
Differences in mRNA expression between control and BAPN-treated samples were assessed
using the REST program in group means for statistical significance by using a Pair Wise Fixed
Allocation Random Test. The peak percentage area ratios for the FTIR experiment, were com-
pared between the control and BAPN groups using a Student’s t-test.
Results
Atomic Force Microscopy
Collagen produced in vitro by MC3T3-E1 preosteoblasts was assessed in 60 mm culture dishes.
5 distinct locations were identified and analyzed in each dish using 3.5 μm x 3.5 μm images
(Fig 2). A minimum of 10 collagen fibrils were imaged from each of 5 locations, amounting to
at least 50 fibrils per dish, and totaling 217 fibril measurements for the control group and 251
for the BAPN-treated group.
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 5 / 13
For each dish, fibril D-spacing measurements were pooled to produce the mean D-spacing
for that dish, and mean values ranged from 65.8 nm to 66.8 nm for the control group and from
66.6 nm to 67.6 nm for the BAPN-treated group. The mean D-spacing for the 4 samples was
66.4 nm ±0.4 nm for the control group and 67.1 nm ±0.4 nm for the BAPN-treated group
(p = 0.060) (Fig 3B). When all fibrils in a group were analyzed together, there was a distribu-
tion of D-spacing values ranging from 60.2 nm to 72.9 nm for the control group and 61.7 to
71.1 nm for the BAPN-treated group (Fig 3A). These distributions were significantly different
from one another with the treated population shifted to higher D-spacing values over most of
its range (A-D test, p<0.0001) (Fig 3B).
Quantitative Reverse Transcription Polymerase Chain Reaction
No significant difference was observed in the mRNA expression of any target gene in the
BAPN-treated samples relative to controls (Table 1).
Fourier Transform Infrared Spectroscopy
Peak fitting resulted in consistent peaks around 1654cm
-1
and 1680cm
-1
as opposed to the
expected 1660cm
-1
and 1690cm
-1
locations. However; a positive spectral shift of ~10cm
-1
would result in positions closely matching those reported elsewhere [32,44], therefore peaks
found in this study are considered to be representative of HP and immature crosslinks (Fig 4).
The spectral shift may be due to interactions with water still present in the samples after air-
drying [45–47]. Treatment with BAPN resulted in a decrease in the collagen crosslink ratio
driven by a significant reduction in the HP crosslink peak percent area (p<0.05). There was
no statistical difference in the percent area of the peak corresponding to immature crosslinks
(Table 2).
Fig 2. Representative 3.5 μm x 3.5 μm collagen AFM image. AFM images of collagen in its native state
were coupled with Fourier transform analysis to measure the periodic fibril D-spacing of collagen synthesized
in vitro by osteoblasts.
doi:10.1371/journal.pone.0166392.g002
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 6 / 13
Discussion
It was hypothesized that BAPN treatment would cause partially differentiated MC3T3-E1 cells
to synthesize a collagen matrix that was morphologically different from that produced by non-
treated cells. Our data indicate that a distribution of D-spacing values, rather than a single
value, exists for Type I collagen produced in vitro by osteoblasts. The results demonstrate that
inhibition of enzymatic crosslinking via BAPN binding of lysyl oxidase causes the D-spacing
distribution to shift towards higher values. D-spacing and its distribution provide information
on the state and internal structure of collagen and can reflect structural defects in its αchains
Fig 3. Collagen D-spacing obtained from the D-spacing measurements in each group (n = 4). A clear
shift towards higher D-spacing values in the BAPN-treated group is evident in the (a) histogram, (b)
cumulative distribution function (CDF), and mean, indicated by the diamond marks on the CDF.
doi:10.1371/journal.pone.0166392.g003
Table 1. Fold changes in mRNA expression of BAPN-treated samples relative to controls (n = 5).
Target Gene Fold Change Std. Error 95% Confidence Interval p-value
COL1A1 1.011 0.759–1.452 0.540–1.562 0.966
COL1A2 1.123 0.787–1.447 0.616–1.738 0.471
LOX 1.094 0.655–1.931 0.448–2.569 0.756
doi:10.1371/journal.pone.0166392.t001
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 7 / 13
or changes due to alterations in post-translational collagen synthesis. The inhibition of enzy-
matic crosslinking via lysyl oxidase inhibition was confirmed by the significant reduction in
the mature/immature crosslink ratio and decrease in the peak percent area corresponding to
HP, as a result of BAPN treatment. While analysis of D-spacing values showed a significant
difference between groups, no difference was found between the control and BAPN-treated
groups when comparing gene expression levels. The data showed that treatment of osteoblasts
with BAPN does not induce a significant change in expression of any of the genes targeted in
Fig 4. Representative mature and immature crosslink peak fittings underneath the FTIR spectral curve. A decrease in the 1654cm
-1
peak area is evident in the BAPN-treated sample relative to control. The BAPN-treated and control samples were plotted on different axes to
visually highlight this difference. The black solid and dashed lines correspond to the full spectra of control and BAPN-treated samples,
respectively.
doi:10.1371/journal.pone.0166392.g004
Table 2. Information on underlying FTIR peaks located at ~1660cm
-1
and ~1690cm
-1
.
Mean Peak Position (cm
-1
) Mean Peak Percent Area Mean Area Ratio
Control ~1660 cm
-1
1654.3730 ±0.7289 16.2868 ±4.1089 3.9068 ±1.6353
~1690 cm
-1
1681.0301 ±1.5651 4.7963 ±2.2037
BAPN ~1660 cm
-1
1653.4087 ±0.9959 8.2149 ±3.4959 1.9865 ±0.6145
~1690 cm
-1
1678.8092 ±1.0640 4.4880 ±2.3100
p-value ~1660 cm
-1
0.0048 0.0338
~1690 cm
-1
0.8177
doi:10.1371/journal.pone.0166392.t002
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 8 / 13
this study for their involvement in collagen synthesis. These results contrast those using higher
BAPN concentrations in which COL1A1 was upregulated [32] and LOX was downregulated
[31,32] with BAPN treatment, and challenges the hypothesis that BAPN would drive upregula-
tion of LOX as a response to the decrease in aldehyde formation. Results of this study highlight
the effects of post-translational collagen modifications on collagen structure while demonstrat-
ing that these changes occurred in the absence of altered collagen or LOX gene expression.
The D-spacing distribution has been shown to be capable of reflecting differences in disease
states, such as osteoporosis [19] and osteogenesis imperfecta [15,18], as well as tissue types,
namely dentin, bone, and tendon [17]. The present study confirms that the D-spacing measure
can capture aspects of collagen fibril structure which may relate to the state and internal struc-
ture of individual molecules. Data revealed a significant upward shift in the D-spacing distri-
bution between control and treatment groups. The difference in mean D-spacing had a
marginally significant p value of 0.06. The low sample size (n = 4) necessitated the use of a non-
parametric statistical test. Given the data from the current study, a post-hoc sample size analy-
sis indicates that a sample size of n = 6 would be needed in order to detect a statistical
difference between the two groups at 80% power.
The introduction of BAPN, and its binding effect on lysyl oxidase, inhibits the enzymatic
crosslinking pathway preventing the formation of aldehyde products. This reduction in alde-
hyde products has been shown in other osteoblast studies to limit the formation of immature
covalent and mature multivalent collagen crosslinks [31,32]. In addition, lysyl oxidase inhibi-
tion by a lower in vivo BAPN-fumarate concentration of 0.025mM was found to cause a signif-
icant shift in D-spacing in mouse bone from another study completed by our group [48]. After
compensating for the added fumarate salt, this concentration corresponds to 0.0137mM
BAPN, as compared with the 0.137mM BAPN concentration used in the current study. Given
that this tenfold higher 0.137mM BAPN concentration corresponded to five times the half-
maximal inhibitory BAPN concentration in vivo [49,50], we expected greater lysyl oxidase
inhibition to be induced in vitro. In other words, the quantitative analysis of collagen synthe-
sized in vitro reflects a direct rather than systemic effect of lysyl oxidase inhibition by BAPN.
The upward shift in D-spacing distribution seen in the BAPN-treated group of this study sug-
gests that crosslinking may be responsible for compression of fibrils under normal conditions.
The same trend was observed in non-mineralized collagen from mouse tail tendon in a study
examining the effect of chemical fixation on mouse tail tendon [51]. In contrast, the D-spacing
distribution was found to shift toward lower values in mineralized mouse bone collagen with
BAPN treatment in vivo [48]. These differences in collagen morphology emphasize the com-
plexity of a crosslink formation process in which in vivo/in vitro lysyl oxidase inhibition, bone/
de novo collagen synthesis, and presence/absence of mineral in the collagen matrix all play a
role in allowing decompression of the collagen fibril.
Quantitative RT-PCR was used to amplify gene sequences found in RNA isolated from con-
trol and BAPN-treated osteoblasts, and to quantify mRNA expression levels. RNA was isolated
from osteoblasts after seven days when collagen mRNA expression and synthesis levels were
highest [52]. When comparing the two groups, the analysis of fold change in expression
showed no difference in LOX,COL1A1, or COL1A2 expression. These data indicate that inhi-
bition of the LOX enzyme by 0.25mM BAPN-fumarate does not drive a significant upregula-
tion of the LOX gene nor does it have a significant effect on the regulation of genes encoding
the α1 and α2 helices that form collagen molecules. This supports other BAPN osteoblast stud-
ies in which genes encoding LOX were not found to be regulated at similar BAPN concentra-
tions [31,32]. This lack of a significant effect suggests that a 0.25mM BAPN-fumarate
concentration is too low to induce osteoblasts to compensate for the lack of aldehyde forma-
tion and, consequently, collagen matrix formation and stabilization.
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 9 / 13
FTIR has been used to obtain information about protein secondary structures [39,40], spe-
cifically as pertaining to enzymatic collagen crosslinks [45,53]. The use of second derivative
methods to locate and fit peaks underlying the FTIR spectral curve has shown a correlation
between peaks at 1660 cm
-1
/1690 cm
-1
and mature/immature crosslinks. [41]. Using this tech-
nique, the current study characterized the secondary structure of collagen synthesized in vitro
by osteoblasts. Peaks were consistently found in the 1654cm
-1
and 1680cm
-1
regions, corre-
sponding to mature HP and immature crosslinks, respectively. BAPN treatment resulted in a
significant reduction in the HP crosslink peak percent area and decrease in the mature to
immature crosslink ratio. The peak percent area of immature crosslinks was not significantly
affected. Collectively, these data suggest that a 0.25mM BAPN-fumarate concentration did not
inhibit the total amount of available lysyl oxidase enzyme because immature crosslinks were
still present. However, the 0.25mM concentration was sufficient to prevent the maturation of
divalent immature crosslinks to trivalent HP crosslinks. Results from this study were similar to
another in which higher BAPN concentrations also caused an HP crosslink decrease [32] and
differed from others which showed no change in the crosslink ratio at comparable BAPN con-
centrations [31]. These differences in the observed effect of lysyl oxidase inhibition on collagen
secondary structure are indicative of a dose-dependent response in enzymatic crosslink forma-
tion for both immature and mature crosslinks, particularly relating to HP.
A more thorough investigation of a dose-dependent response to BAPN would elucidate the
effect of lysyl oxidase inhibition on collagen gene expression and the single dosage used is con-
sidered a limitation of the present study. Consideration of other LOX isoforms such as LOXL1-
4could also provide valuable information in the context of this study given that their expres-
sion could differ from that of LOX. Future studies will be aimed at assessing these questions
and how they relate to osteoblast signaling, collagen production, and post-translational
modification.
Conclusions
In conclusion, collagen synthesized in vitro by pre-osteoblastic cells was found to be fibrillar
and organized in a manner to produce natural variation in its periodic D-spacing. Although
there were no differences in the expression of genes relating to collagen synthesis or enzymatic
crosslink initiation, partial lysyl oxidase inhibition at low levels of BAPN still resulted in signif-
icant morphological and crosslinking changes in collagen. Collagen fibrils of the BAPN-treated
group were found to be morphologically different from those of the control group as seen by
the significant increase in the D-spacing distribution of the BAPN-treated collagen fibrils. In
addition, the ratio of mature to immature crosslinks was found to decrease with BAPN treat-
ment, associated with a reduction in peak percent area of mature crosslink HP. These findings
were made possible by in vitro treatment with BAPN and analysis of collagen synthesized
entirely under direct inhibition of lysyl oxidase and, thus, enzymatic crosslink formation.
Acknowledgments
The authors are grateful to the IUPUI Chemistry Department, particularly Cary Pritchard, for
providing access to the Nicolet iS10 spectrometer and sample preparation guidance. The
authors wish to confirm that there are no potential conflicts of interest with respect to the
authorship and/or publication of this article.
Author Contributions
Conceptualization: SPC JMW.
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 10 / 13
Data curation: SPC JMW.
Formal analysis: SPC JMW.
Funding acquisition: JMW.
Investigation: SPC JMW.
Methodology: SPC JMW.
Project administration: SPC JMW.
Resources: SPC JMW.
Supervision: SPC JMW.
Validation: SPC JMW.
Visualization: SPC JMW.
Writing – original draft: SPC JMW.
Writing – review & editing: SPC JMW.
References
1. Burr DB, Allen MR. Basic and Applied Bone Biology. 1st ed. Burr D, Allen M, editors. San Diego, CA:
Elsevier; 2013.
2. Boskey A, Wright T, Blank R. Collagen and bone strength. J bone Miner Res. 1999; 14: 330–335. Avail-
able: http://onlinelibrary.wiley.com/doi/10.1359/jbmr.1999.14.3.330/full doi: 10.1359/jbmr.1999.14.3.
330 PMID: 10027897
3. Burr D. The contribution of the organic matrix to bone’s material properties. Bone. 2002; 31: 8–11. doi:
10.1016/S8756-3282(02)00815-3 PMID: 12110405
4. Viguet-Carrin S, Garnero P, Delmas PD. The role of collagen in bone strength. Osteoporos Int. 2006;
17: 319–36. doi: 10.1007/s00198-005-2035-9 PMID: 16341622
5. Cummings SR, Kelsey JL, Nevitt MC, O’Dowd KJ. Epidemiology of osteoporosis and osteoporotic frac-
tures. Epidemiol Rev. 1985; 7.
6. Becker S, Ogon M. Epidemiology of osteoporosis. Balloon Kyphoplasty. 2008; 16: 1–3. doi: 10.1007/
978-3-211-74221-1_1
7. WHO. Assessment of fracture risk and its application to screening for postmenopausal osteoporosis:
Report of a WHO study group. World Health Organ Tech Rep Ser. 1994; 843. doi: 10.1007/
BF01622200 PMID: 7941614
8. Rauch F, Glorieux FH. Osteogenesis imperfecta. Lancet. 2004; 363: 1377–85. doi: 10.1016/S0140-
6736(04)16051-0 PMID: 15110498
9. Jepsen KJ, Schaffler MB, Kuhn JL, Goulet RW, Bonadio J, Goldstein S a. Type I collagen mutation
alters the strength and fatigue behavior of Mov13 cortical tissue. J Biomech. 1997; 30: 1141–7. Avail-
able: http://www.ncbi.nlm.nih.gov/pubmed/9456382 PMID: 9456382
10. Byers PH, Wallis G a, Willing MC. Osteogenesis imperfecta: translation of mutation to phenotype. J
Med Genet. 1991; 28: 433–442. doi: 10.1136/jmg.28.7.433 PMID: 1895312
11. Ottani V, Martini D, Franchi M, Ruggeri A, Raspanti M. Hierarchical structures in fibrillar collagens.
Micron. 2002; 33: 587–596. Available: http://www.sciencedirect.com.ezproxy.lib.purdue.edu/science/
article/pii/S0968432802000331 PMID: 12475555
12. Kadler KE, Holmes DF, Trotter JA, Chapman JA. Collagen fibril formation. Biochem J. 1996; 316 (Pt 1:
1–11. Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1217307&tool=
pmcentrez&rendertype=abstract
13. Hulmes DJS, Miller A, Parry DAD, Piez KA, Woodhead-Galloway J. Analysis of the primary structure of
collagen for the origins of molecular packing. J Mol Biol. 1973; 79: 137–148. Available: http://www.
sciencedirect.com/science/article/pii/0022283673902751 PMID: 4745843
14. Hodge AJ, Petruska J. Recent studies with the electron microscope on ordered aggregates of the tropo-
collagen molecule. In: Ramachandran GM, editor. Aspects of protein structure. 1963. p. 289.
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 11 / 13
15. Kemp AD, Harding CC, Cabral WA, Marini JC, Wallace JM. Effects of tissue hydration on nanoscale
structural morphology and mechanics of individual Type I collagen fibrils in the Brtl mouse model of
Osteogenesis Imperfecta. J Struct Biol. Elsevier Inc.; 2012; 180: 428–38. doi: 10.1016/j.jsb.2012.09.
012 PMID: 23041293
16. Erickson B, Fang M, Wallace JM, Orr BG, Les CM, Banaszak Holl MM. Nanoscale structure of type I
collagen fibrils: quantitative measurement of D-spacing. Biotechnol J. 2013; 8: 117–26. doi: 10.1002/
biot.201200174 PMID: 23027700
17. Wallace JM, Chen Q, Fang M, Erickson B, Orr BG, Banaszak Holl MM. Type I collagen exists as a distri-
bution of nanoscale morphologies in teeth, bones, and tendons. Langmuir. 2010; 26: 7349–54. doi: 10.
1021/la100006a PMID: 20121266
18. Wallace JM, Orr BG, Marini JC, Holl MMB. Nanoscale morphology of Type I collagen is altered in the
Brtl mouse model of Osteogenesis Imperfecta. J Struct Biol. Elsevier Inc.; 2011; 173: 146–52. doi: 10.
1016/j.jsb.2010.08.003 PMID: 20696252
19. Wallace JM, Erickson B, Les CM, Orr BG, Banaszak Holl MM. Distribution of type I collagen morpholo-
gies in bone: relation to estrogen depletion. Bone. Elsevier Inc.; 2010; 46: 1349–54. doi: 10.1016/j.
bone.2009.11.020 PMID: 19932773
20. Avery NC, Sims TJ, Bailey AJ. Quantitative determination of collagen cross-links. Methods Mol Biol.
2009; 522: 103–21. doi: 10.1007/978-1-59745-413-1_6 PMID: 19247601
21. Eyre DR, Paz M a, Gallop PM. Cross-linking in collagen and elastin. Annu Rev Biochem. 1984; 53:
717–48. doi: 10.1146/annurev.bi.53.070184.003441 PMID: 6148038
22. Eyre DR, Wu J-J. Collagen Cross-Links. Top Curr Chem. Springer Berlin Heidelberg; 2005; 247: 207–
229. doi: 10.1007/b103828
23. Saito M, Marumo K. Collagen cross-links as a determinant of bone quality: a possible explanation for
bone fragility in aging, osteoporosis, and diabetes mellitus. Osteoporos Int. 2010; 21: 195–214. doi: 10.
1007/s00198-009-1066-z PMID: 19760059
24. Dasler W. Isolation of toxic crystals from sweet peas (Lathyrus odoratus). Science (80-). 1954; 120:
307–308. Available: http://www.sciencemag.org/content/120/3112/307.short PMID: 13186839
25. Nimni ME. Mechanism of inhibition of collagen crosslinking by penicillamine. Proc R Soc Med. Royal
Society of Medicine Press; 1977; 70 Suppl 3: 65–72. Available: http://www-ncbi-nlm-nih-gov.proxy.
medlib.iupui.edu/pmc/articles/PMC1543564/
26. Siegel RC. Collagen cross-linking. Effect of D-penicillamine on cross-linking in vitro. J Biol Chem. 1977;
252: 254–9. Available: http://www.ncbi.nlm.nih.gov/pubmed/13066 PMID: 13066
27. McNerny EMB, Gong B, Morris MD, Kohn DH. Bone fracture toughness and strength correlate with col-
lagen cross-link maturity in a dose-controlled lathyrism mouse model. J Bone Miner Res. 2015; 30:
455–464. doi: 10.1002/jbmr.2356 PMID: 25213475
28. Lees S, Hanson D, Page E, Mook HA. Comparison of dosage-dependent effects of beta-aminopropioni-
trile, sodium fluoride, and hydrocortisone on selected physical properties of cortical bone. J bone Miner
Res. 1994; 9: 1377–89. doi: 10.1002/jbmr.5650090909 PMID: 7817821
29. Paschalis EP, Tatakis DN, Robins S, Fratzl P, Manjubala I, Zoehrer R, et al. Lathyrism-induced alter-
ations in collagen cross-links influence the mechanical properties of bone material without affecting the
mineral. Bone. Elsevier Inc.; 2011; 49: 1232–1241. doi: 10.1016/j.bone.2011.08.027 PMID: 21920485
30. Fernandes H. The role of collagen crosslinking in differentiation of human mesenchymal stem cells and
MC3T3-E1 cells. Tissue Eng Part A. 2009; 15. Available: http://online.liebertpub.com/doi/abs/10.1089/
ten.tea.2009.0011
31. Thaler R, Spitzer S, Rumpler M, Fratzl-Zelman N, Klaushofer K, Paschalis EP, et al. Differential effects
of homocysteine and beta aminopropionitrile on preosteoblastic MC3T3-E1 cells. Bone. 2010; 46: 703–
709. doi: 10.1016/j.bone.2009.10.038 PMID: 19895920
32. Turecek C, Fratzl-Zelman N, Rumpler M, Buchinger B, Spitzer S, Zoehrer R, et al. Collagen cross-link-
ing influences osteoblastic differentiation. Calcif Tissue Int. 2008; 82: 392–400. doi: 10.1007/s00223-
008-9136-3 PMID: 18488133
33. Fernandes H, Dechering K, Van Someren E, Van Blitterswijk C, de Boer J. Investigating the role of the
extracellular matrix on differentiation of human mesenchymal stem cells and MC3T3 cells. Bone. 2008.
p. S21. doi: 10.1016/j.bone.2007.12.020
34. Gonzalez AD, Gallant MA, Burr DB, Wallace JM. Multiscale analysis of morphology and mechanics in
tail tendon from the ZDSD rat model of type 2 diabetes. J Biomech. 2014; 47: 681–6. doi: 10.1016/j.
jbiomech.2013.11.045 PMID: 24360194
35. Wallace JM, Golcuk K, Morris MD, Kohn DH. Inbred strain-specific response to biglycan deficiency in
the cortical bone of C57BL6/129 and C3H/He mice. J bone Miner Res. 2009; 24: 1002–12. doi: 10.
1359/jbmr.081259 PMID: 19113913
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 12 / 13
36. Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: vali-
dation by real-time, quantitative RT-PCR. J Biochem Biophys Methods. 2000; 46: 69–81. doi: 10.1016/
S0165-022X(00)00129-9 PMID: 11086195
37. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids
Res. 2001; 29: 45e–45. doi: 10.1093/nar/29.9.e45
38. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST(C)) for group-wise com-
parison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002;
30: 36e–36. doi: 10.1093/nar/30.9.e36
39. Yang H, Yang S, Kong J, Dong A, Yu S. Obtaining information about protein secondary structures in
aqueous solution using Fourier transform IR spectroscopy. Nat Protoc. 2015; 10: 382–396. doi: 10.
1038/nprot.2015.024 PMID: 25654756
40. Dong a, Huang P, Caughey WS. Protein secondary structures in water from second-derivative amide I
infrared spectra. Biochemistry. 1990; 29: 3303–3308. doi: 10.1021/bi00465a022 PMID: 2159334
41. Paschalis EP, Verdelis K, Doty SB, Boskey AL, Mendelsohn R, Yamauchi M. Spectroscopic characteri-
zation of collagen cross-links in bone. J bone Miner Res. 2001; 16: 1821–8. doi: 10.1359/jbmr.2001.16.
10.1821 PMID: 11585346
42. Paschalis EP, Gamsjaeger S, Tatakis DN, Hassler N, Robins SP, Klaushofer K. Fourier Transform
Infrared Spectroscopic Characterization of Mineralizing Type I Collagen Enzymatic Trivalent Cross-
Links. Calcif Tissue Int. 2014; doi: 10.1007/s00223-014-9933-9 PMID: 25424977
43. Hammond MA, Berman AG, Pacheco-Costa R, Davis HM, Plotkin LI, Wallace JM. Removing or truncat-
ing connexin 43 in murine osteocytes alters cortical geometry, nanoscale morphology, and tissue
mechanics in the tibia. Bone. 2016; 88: 85–91. doi: 10.1016/j.bone.2016.04.021 PMID: 27113527
44. Varga F, Rumpler M, Zoehrer R, Turecek C, Spitzer S, Thaler R, et al. T3 affects expression of collagen
I and collagen cross-linking in bone cell cultures. Biochem Biophys Res Commun. 2010; 402:180–5.
doi: 10.1016/j.bbrc.2010.08.022 PMID: 20707983
45. Farlay D, Duclos M-E, Gineyts E, Bertholon C, Viguet-Carrin S, Nallala J, et al. The ratio 1660/1690 cm
(-1) measured by infrared microspectroscopy is not specific of enzymatic collagen cross-links in bone
tissue. Laird EG, editor. PLoS One. Public Library of Science; 2011; 6. doi: 10.1371/journal.pone.
0028736 PMID: 22194900
46. Byler DM, Susi H. Examination of the Secondary Structure of Proteins by Deconvolved FTIR Spectra.
10.1002/bip.360250307
47. Lazarev YA, Lazareva A V. Infrared Spectra and Structure of Synthetic Polytripeptides.
48. Hammond M a, Wallace JM. Exercise prevents β-aminopropionitrile-induced morphological changes to
type I collagen in murine bone. Bonekey Rep. 2015; 4: 645. doi: 10.1038/bonekey.2015.12 PMID:
25798234
49. Nagan N, Callery PS, Kagan HM. Aminoalkylaziridines as substrates and inhibitors of lysyl oxidase:
specific inactivation of the enzyme by N-(5-aminopentyl)aziridine. Front Biosci. 1998; 3: A23–A26.
PMID: 9563974
50. Trackman PC, Kagan HM. Nonpeptidyl amine inhibitors are substrates of lysyl oxidase. J Biol Chem.
1979; 254: 7831–7836. PMID: 38246
51. Wallace JM. Effects of fixation and demineralization on bone collagen D-spacing as analyzed by atomic
force microscopy. Connect Tissue Res. Informa Healthcare USA, Inc. New York; 2015; 1–8. doi: 10.
3109/03008207.2015.1005209 PMID: 25634588
52. Gerstenfeld LC, Chipman SD, Kelly CM, Hodgens KJ, Lee DD, Landis WJ. Collagen expression, ultra-
structural assembly, and mineralization in cultures of chicken embryo osteoblasts. J Cell Biol. 1988;
106: 979–89. Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2115070&tool=
pmcentrez&rendertype=abstract PMID: 3346332
53. Paschalis EP, Shane E, Lyritis G, Skarantavos G, Mendelsohn R, Boskey AL. Bone fragility and colla-
gen cross-links. J bone Miner Res. 2004; 19: 2000–4. doi: 10.1359/JBMR.040820 PMID: 15537443
β-Aminopropionitrile Effect on Collagen Morphology and Molecular Composition In Vitro
PLOS ONE | DOI:10.1371/journal.pone.0166392 November 9, 2016 13 / 13
