International Journal of Biological Macromolecules 97 (2017) 5566

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Collagen type I from bovine bone. Effect of animal age, bone anatomy
and drying methodology on extraction yield, self-assembly, thermal
behaviour and electrokinetic potential
Vincenza Ferraro a,b, , Brigitte Gaillard-Martinie c , Thierry Sayd a , Christophe Chambon a ,
Marc Anton b , Vronique Sant-Lhoutellier a
a
UR 370 QuaPA (Qualit de Produits Animaux) INRA, 63122 Saint-Gens-Champanelle, France
b
UR 1268 BIA (Biopolymres, Interactions, Assemblages) INRA, 44300 Nantes, France
c
UR 454 Microbiologie INRA, 63122 Saint-Gens-Champanelle, France

a r t i c l e i n f o a b s t r a c t

Article history: Natural collagen is easily available from animal tissues such as bones. Main limitations reported in the
Received 20 September 2016 use of natural collagen are heterogeneity and loss of integrity during recovery. However, its natural
Received in revised form complexity, functionality and bioactivity still remain to be achieved through synthetic and recombinant
10 December 2016
ways.
Accepted 21 December 2016
Variability of physicochemical properties of collagen extracted from bovine bone by acetic acid was
Available online 27 December 2016
then investigated taking into account endogenous and exogenous factors. Endogenous: bovines bones
age (4 and 7 years) and anatomy (femur and tibia); exogenous: thermal treatments (spray-drying and
Keywords:
Bone
lyophilisation). Scanning electron microscopy, spectroscopy (EDS, FTIR, UV/Vis and CD), differential scan-
Collagen ning calorimetry (DSC), centesimal composition, mass spectrometry, amino acids and zeta-potential
Extraction analysis were used for the purpose.
Protein Age correlated negatively with yield of recovery and positively with minerals and proteoglycans con-
Self-assembly tent. Comparing the anatomy, higher yields were found for tibias, and higher stability of tibias collagen
in solution was noticed. Whatever the age and the anatomy, collagens were able to renature and to self-
assemble into tri-dimensional structures. Nonetheless thermal stability and kinetics of renaturation were
different.
Variability of natural collagen with bone age and anatomy, and drying methodology, may be a crucial
advantage to conceive tailor-made applications in either the biological or technical sector.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Collagens are a large family of proteins with a brous, hierarchi-
cal nature endowed by a triple-helical supramolecular structure.
They represent ca. 30% of the total protein pool in vertebrates, and
their size, function and distribution in the extracellular matrix of
tissues varies considerably [1,4]. Collagens main role is to provide
structure and mechanical properties to vertebrates by the arrange-
ment into insoluble brils with high strength. However, collagens
also play important biological functions in cellular processes such
as signaling, differentiation, motion, communication and apoptosis.
Moreover, collagens are able to bind to growth-factors promoting
tissues repair and regeneration [1,5,6].
Corresponding author at: UR 370 QuaPA (Qualit de Produits Animaux) INRA,
63122 Saint-Gens-Champanelle, France.
E-mail address: vincenza.ferraro@inra.fr (V. Ferraro).
Among the 29 distinct types of collagens identied till now, type
I is the most abundant. It belongs to the class of brils-forming
collagens (also including type II, III, IV, XI, XXIV and XXVII), which
represent 90% of the whole collagens family. Type I collagen consti-
tutes 9095% of organic mass of bone and is the major component
of tendons, skin, ligaments and cornea. Tensile strength, torsional
stiffness and load bearing in vertebrates are endorsed by collagen
type I [1,4,7]. Collagen is among the rst proteins to be known, how-
ever it has started to be intensively studied only back in 1940 and
the structure of the triple-helix has been conrmed for the rst time
in 1994 by X-rays crystallography. The quasi-hexagonal arrange-
ment of microbrils, the location of telopeptides and the covalent
cross-linking interaction of tropocollagen monomers to each other
has been elucidated only in 2001 and much on self-assembly is still
unclear [2,6].
Collagen type I is promptly available through leaching from ani-
mal tissues, namely skin, tendons and bones which are normally

http://dx.doi.org/10.1016/j.ijbiomac.2016.12.068
0141-8130/ 2016 Elsevier B.V. All rights reserved.
56 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566
discarded during slaughtering [6,8]. High quantities of residues
are generated throughout land and aquatic animal processing for
human consumption; it is estimated that 20 and 100 tons of dis-
cards are generated worldwide from sheries and meat (cattle,
pigs and poultry) respectively, where ca. 5.2 and 16.5 million
tons, respectively, arise from the sole European Union [8]. Differ-
ent chemical agents and pre-treatments can be used to extract
collagen and each method leads to a collagen molecule with dif-
ferent biological, chemical and morphological properties [2,6,9].
Salt can solubilize not cross-linked collagens (i.e. newly synthe-
sized collagen) however it results in low yield and purity. Organic
acids can solubilize non-cross-linked collagens and can also break
some of interchain cross-linkages (between the three chains of the
tropocollagen molecule), such as the reducible aldimine condensa-
tion (Shift base) cross-links. Enzymes, such as pepsin, increase the
yield of collagen; however, while the triple-helical structure is still
preserved, proteolysis to telopeptides occurs with implication in
ex vivo self-assembly [3,6]. In fact, although collagen telopeptides
might not be essential for brillogenesis of collagen, their absence
greatly weakens the mature bril due to the lack of cross-links
within and between triple-helices [2].
Collagen recovered from animal sources could be efciently
used in many domains such as biomedicine, biomaterials, cos-
metics, textiles, development of environmental-friendly materials
[3,6,8]. Although some limitations are present, like heterogeneity,
possible immunogenicity and loss of structural integrity during the
isolation process, natural collagen still presents important advan-
tages with respect to recombinant or synthetic analogues [1,3,6].
The incorporation of post-translational modications, such as gly-
cosylation, hydroxylation of proline residues and cross-linking of
tropocollagen monomers, is in fact very difcult in the heterologous
production of collagen and would need complex expression sys-
tems. On the other hand, synthetic collagen-like proteins and brils
lack many of the characteristics of higher-ordered natural colla-
gen structures. Synthetic collagens that closely mimic the length,
girth, patterns, mechanical properties and complexity of natural
collagen brils remain to be developed even though some progress
have been made [2,6]. But principally, still remain the character-
istic of natural collagen to interact with many other proteins and
biomolecules, a crucial future on which relies its bioactivity and the
possibility of treatment of diseases [1,3,6].
Modications in collagen cross-linking with age is known [2,3,6]
however, few and quite old studies are available on the effect of
age and anatomy on collagen solubility in acetic acid [10,11] and
most of the data are referred to skin and tendon [11,13]. Rela-
tionship between thermal treatment and bovine type I collagen
self-assembly is known and it currently exploited for different
industrial application [14,15]. Effects of thermal treatment on the
properties of rehydrated collagen have been also reported by some
authors [16]. However, no data are available on the possible hetero-
geneity of type I bovine bone collagen self-assembly and stability
in solution according to age, anatomy and drying methodology.
This research aimed to investigate the effect of endogenous and
exogenous factors on the variability of physicochemical properties
of natural collagen type I recovered from bovine bones by acetic
acid. The age 4 and 7 years old and the anatomy of cows
leg bones femur and tibia have been taken into account as
endogenous parameters. As exogenous factors, two thermal treat-
ments spray drying and lyophilisation have been considered to
stabilize collagen after acid extraction. Therefore, yields of recov-
ery, minerals and proteoglycans concentration, thermal stability,
renaturation and heat effects on collagen self-assembly, accord-
ing to bone age and anatomy, have been investigated by several
techniques.
2. Materials and methods
2.1. Samples
Cortical section of legs bones femur and tibia from a young
and an old milk cow (4 and 7 years respectively), Holstein breed, has
been collected at the experimental slaughterhouse of INRA, QuaPA
(Quality of Animal Products) Unit in Clermont-Ferrand (France).
Bones have been identied as follows: FOB for old femur (7 years
old), FYB for young femur (4 years old), TOB for old tibia (7 years
old) and TYB for young tibia (4 years old). After collection, bones
have been stored at 30 C until processed for the extraction of
collagen.
2.2. Extraction of collagen
Collagen has been recovered from bones by a mild acid treat-
ment in order to maintain intact the structure of tropocollagen
chains [12]. Prior to extraction, bones have been cut into slices of
1 cm thick and then in pieces of approximately 1 1 cm. The mate-
rial has been washed with water in order to remove impurities,
then soaked in 90% (v/v) ethanol (Sigma-Aldrich; Saint-Quentin-
Fallavier, France) for 15 min to remove fat traces and microbial
contamination due to handling, and washed with water again to
remove ethanol. Afterwards, bones have been dried at 40 C for
1 day, and nally ground in a Gondard hammer miller, model BJL
8500-02 (Boisson Jean-Luc; Lusignan, France) to favour the extrac-
tion of collagen. To extract collagen, bone powders have been
soaked in 0.5 M acetic acid (Sigma-Aldrich) for 5 days at 4 C, at
a ratio bone:acetic acid solution 1:5 (w/v). The mixtures have been
stirred from time to time to favour acetic acid diffusion into bone
powders. At the end of extraction, mixtures have been centrifuged
at 4000g and 4 C for 30 min, and collagen solutions have been
recovered as the supernatant Collagen solutions (extracts) have
been identied as FOS, FYS, TOS and TYS.
2.3. Drying of collagen solutions
Collagen solutions have been dried by two different method-
ologies, namely spray-drying and lyophilisation. Spray-drying has
been accomplished by the BCHI B-290 equipment (BCHI; Rungis,
France) at input and output temperatures of 150 and 70 C respec-
tively and at a ow-rate of 2 mL/min. Lyophilisation has been
carried out by the equipment Cryotec LPCCPLS15 (Cryotec; Saint-
Gly-du-Fesc, France) after collagen solutions freezing at 30 C.
Collagen powders have been identied as follows: FOSD, TOSD,
FYSD and TYSD in the case of spray-drying, and FOL, TOL, FYL and
TYL for lyophilisation.
2.4. Centesimal composition free water, ash and organic matter
content
Free water content in milled bones and in collagen powders after
drying has been determined by heating at 102 C for 48, according
to EN 12880:2000 [17]. Acetic acids residue in collagen powders
has been determined thereafter by drying at 130 C for 48 h, taking
into account the acetic acid evaporating temperature of 118 C. Ash
content in milled bones and in collagen powders has been assessed
by calcination at 550 C overnight according to the AOACs Ofcial
methods of analysis [18]. Organic matter has been determined by
subtracting amount of humidity, acetic acid and ash to the total
matter. All determinations have been done in triplicate.
 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566
2.5. Protein content
Protein content in bones powders has been determined by the
Kjeldahl method [19]. A nitrogen-to-protein conversion factor of
5.20 has been used taking into account that nitrogen content in col-
lagen is 19.2% w/w, instead of 16% w/w as in food proteins [20,21].
All determinations have been done in triplicate.
2.6. Carbon content
Total carbon in collagen powders has been determined by ash
combustion [22] with the equipment Vario ISOTOPE cube (Elemen-
tar; Lyon, France) and by spectroscopy as reported in Section 2.11.
2.7. Calcium content
Calcium in collagen powders has been analysed by ion chro-
matography, with the equipment Metrohm 850 Professional IC
(Metrohm; Villebon-sur-Yvette, France). The ion exchange resin
column Metrosep C4 150 4 mm has been used as a stationary
phase while mobile phase has been 1.7 mM nitric acid and 0.7 mM
dipicolinic acid (Sigma-Aldrich) in ultra-pure water. Detection has
been done by conductivity. Calcium has been also analysed by spec-
troscopy as reported in Section 2.11.
2.8. Proteoglycans content by glucose determination
Proteoglycans content in collagen powders has been esti-
mated by quantication of total glucose with the phenol-sulphuric
method reported by DuBois et al. [23]. Powders have been dissolved
in 50 mM acetic acid at a concentration of 100 mg/mL. Determina-
tion has been done in triplicate.
2.9. Amino acids analysis
Amino acids analysis in collagen powders has been performed
with the Hitachi L-8900 analyser (Hitachi; Bron, France) with nin-
hydrin post-column derivatisation and detection in the visible
region. Acid hydrolysis with 6 N HCl (Sigma-Aldrich) at 110 C has
been carried out over 24 h. For tryptophan and sulphur contain-
ing amino acids determination samples have been oxidized with
performic acid at 50 C for 10 min prior to acid hydrolysis.
2.10. Proteins identication by mass spectrometry
Collagen identication has been performed by liquid chro-
matography coupled to tandem mass spectrometry (LCMS/MS).
Each collagen sample has been hydrolysed with the enzyme col-
lagenase B (Sigma-Aldrich) with the ratio collagen:collagenase
20:1 (w/w). Hydrolysis has been performed at the optimal con-
ditions of 37 C and pH 7 over 24 h. Peptides mixtures have been
then analysed by a nano-LCMS/MS using an Ultimate 3000 sys-
tem (Dionex; Voisins le Bretonneux, France) coupled to an LTQ
velos Orbitrap (ThermoFisher Scientic) equipped with a nano ion
source. Two microliters of each hydrolysate have been loaded on a
desalting C18 pre-column (500 m inner diameter 5 mm lenght;
Dionex) at 40 L/min in 99.9% H2 O and 0.1% triuoroacetic acid
(Sigma-Aldrich). After 6 min of desalting, the pre-column has been
switched on line with the analytical C18 Pepmap column (75 m
inner diameter 15 cm lenght) equilibrated in 98% solvent A (99.9%
H2 O, 0.1% formic acid) and 2% solvent B (99.9% acetonitrile, 0.1%
formic acid). Peptides have been eluted using a gradient of 230%
solvent B during 35 min at a 350 nL/min ow rate.
Protein identication has been obtained by the Mascot soft-
ware (v2.51, www.matrixscience.com) and Bos taurus database
57
(2015/08, 19840 sequence, www.uniprot.org). Peptide mass toler-
ance has been set to 15 ppm and fragment mass tolerance to 0.5 Da.
Protein identication has been considered valid if at least two pep-
tides with a statistically signicant Mascot score have been found
(False Discovery Rate (FDR) at 5%). Only identications of collagen
and the proteoglycans decorin and biglycan have been reported in
the results.
2.11. Microstructural analysis
2.11.1. Field emission gun scanning electron microscopy
(FEG-SEM)
Bones before collagen extraction have been observed with the
equipment Quanta FEG (Field Emission Gun) 250 (FEI Company,
Mrignac, France) at the accelerating voltage of 15 kV and mag-
nication 14000. Bone samples have been sputter-coated with
carbon.
2.11.2. Scanning electron microscopy energy dispersive X-ray
spectrometry (SEM-EDS)
SEM-EDS has been performed on collagen powders to determine
self-assembly structure and punctual elementary composition in
term of carbon, calcium and phosphorous. The equipment JEOL
JSM-6330F coupled with a QUANTAX energy dispersive X-ray spec-
trometer (BRUKER; Marne la Valle, France) has been used at
the accelerating voltage 515 kV and in the photon energy range
110 keV. Samples have been sputter-coated with gold and the
signal of elements has been recorder on ve points.
2.12. Differential scanning calorimetry (DSC)
Differential scanning calorimetry has been carried out with the
equipment DSC Q2000 TA Instruments (Paris, France). The analysis
has been performed for milled bones and for collagen powders,
kept at 40 C overnight before the essay. An amount of 46 mg has
been placed into hermetically sealed aluminum pans. Two heating
cycles on each sample have been performed: one cycle from 10 C
to 50 C at 1 C/min heating rate, and another cycle from 10 C to
275 C at 10 C/min heating rate. Analysis has been carried out in
duplicate.
2.13. Attenuated total reectance Fourier transform infrared
spectroscopy (ATR-FTIR)
FTIR spectra of milled bones and collagen powders have been
collected in the mid infrared (MIR) region between 4000 and
400 cm1 . The instrument BRUKER Vertex 70 series (BRUKER)
equipped with attenuated total reectance cell has been used. Spec-
tra have been collected as a result of 60 scans each and at 4 cm1
resolution.
2.14. UV/vis absorption spectra
Spectra in the ultraviolet and visible wavelength range have
been collected by the Jasco V-770 spectrophotometer (JASKO; Lyon,
France). Collagen powders have been dissolved in 50 mM acetic
acid at a concentration of 3 mg/mL and have been compared with
collagen from for rat tail at 1 mg/mL.
2.15. Circular dichroism (CD) spectroscopy
CD spectroscopy has been carried out for collagen powders.
Samples have been dissolved in 50 mM acetic acid at the concen-
tration of 1% w/v and dialysed against 200 vols of same solution
through 6 kDa cut-off. Collagen concentration in solution after dial-
ysis has been veried with the Pierce BCA assay (Sigma-Aldrich)
58 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566
Fig. 1. Quanta FEG micrographs of bones powders.
using the JASKO V-770 spectrophotometer (JASKO; Lyon, France).
The color yield has been standardised against a solution of rat
tail collagen (Sigma-Aldrich) in the range of linearity 5250 g/mL
50 mM acetic acid. All determinations have been done in trip-
licate. Before CD, each sample solution has been aliquoted in
three fractions stored at the ambient temperature, at the refrig-
erating temperature 4 C and at the freezing temperature 20 C,
respectively, for 5 days. The spectropolarimeter equipment JASKO
J-810 has been used and the far ultra-violet wavelength range
190260 nm has been analysed.
2.16. Zeta potential of collagen in solution
Zeta potential () has been estimated for collagen powders
obtained by spray-drying and lyophilisation in order to assess the
effect of heat on collagen stability in solution, according to bone age
and anatomy. Samples have been dialysed and ltered as reported
for CD spectroscopy. The equipment Delsa Nano Submicron (Beck-
man Coulter; Villepinte, France) has been used. Analysis has been
performed in triplicate at 10 scans for each measure.
2.17. Statistical analysis
Analysis of variance (ANOVA) has been carried out with the soft-
ware STATISTICA v.12 setting a condence level of 95% (p 0.05).
Principal components analysis (PCA) on the variables considered
for each collagen powder has been performed.
3. Results and discussion
3.1. Collagen microstructure and macroscopic composition
Bone structure and composition, either at macroscopic or micro-
scopic level, is not constant with age and anatomy [24]. In bone, as
well as in many tissues, collagen brils inside bres are oriented not
only longitudinally but also horizontally and transversally; bres
are not just parallel but cross each other and form spirals in order
to increase bone strength [4,25]. For the bovine bones considered,
FEG-SEM micrographs (Fig. 1) showed that collagen bres are thin-
ner in tibia, either old or young, and that brils in young tibia are
not uniform in diameter. Dimension of collagen bres is regulated
by a physical equilibrium between the growing insoluble brils
and the soluble procollagen (precursor of tropocollagen). The pep-
tides termini in procollagen play a crucial role in bre development
where the C-propeptides assure procollagen solubility while the N-
propeptides control the brils shape [4]. As reported in literature
[24], distribution and size of mineral crystals also varies with age
and anatomy. This phenomenon can be noticed in old femur (Fig. 1)
where collagen bres (and brils within bres) are less tight due
to the increased size and displacement of mineral crystals.
 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566 59

Fig. 2. SEM micrographs of spray-dried (left) and lyophilized (right) collagen solutions.

These age/anatomy related modications in the bone matrix FO < TO < FY < TY (Table 1). Yield has been expressed as the quantity
have been reected on the yield of collagen recovered and on its of organic matter recovered through acetic acid with respect to the
physicochemical properties. According to the centesimal composi- organic matter present in the bone, as follows:
tion reported in Table 1, amount of organic matter and minerals is
comparable among bones and it is in agreement with values found
in literature [4,24,26]. Nonetheless, collagen yield has been signif- me OMe DMe
icantly different among bones (p > 0.05) and varied in the order YC (%dw) = 100 (1)
mB OMB DMB
60 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566

Table 1
Centesimal composition (mean value standard deviation, n = 3) of bones and collagens powders; zeta potential () of collagen powders in 50 mM acetic acid; carbon, calcium,
phosphorous and glucose content, and yield of collagen.

FOB TOB FYB TYB

Dry matter (% w/w) 89.2 0.3 89.3 0.2 89.4 0.5 89.5 0.2
Free water (% w/w) 10.8 0.1 10.7 0.2 10.6 0.1 10.5 0.1
Organic matter (% dw) 30.1 0.5 31.0 0.3 30.7 0.5 29.2 0.3
Kjeldahl protein (% dw) 29.5 0.2 30.3 0.3 29.8 0.3 28.5 0.2
Mineral matter (% dw) 69.9 0.3 69 0.4 69.3 0.3 70.8 0.5
Granulometry (mm) 1.78 1.32 1.15 1.15 1.23 1.11 0.88 1.22

FOSD TOSD FYSD TYSD

Dry matter (% w/w) 90.48 0.1 88.9 0.2 90.2 0.2 91.11 0.1
Free water (% w/w) 5.6 0.2 8.3 0.3 7.4 0.3 7.1 0.1
Acetic acid (% w/w) 3.9 0.3 2.7 0.2 2.4 0.1 1.8 0.1
Organic matter (% dw) 35.3 0.3 35.7 0.2 39.0 0.1 39.9 0.2
Mineral matter (% dw) 64.7 0.2 64.5 0.1 61.1 0.2 60.1 0.2
(mV) 12.95 0.4 40.28 0.7 13.96 0.5 38.07 0.6

FOL TOL FYL TYL

Dry matter (% w/w) 95.3 0.2 95.8 0.1 94.8 0.3 95.1 0.2
Free water (% w/w) 3.2 0.1 2.9 0.2 3.4 0.1 3.3 0.1
Acetic acid (% w/w) 1.7 0.1 1.3 0.3 1.8 0.2 1.6 0.1
Organic matter (% dw) 35.3 0.3 35.7 0.2 39.0 0.1 40.0 0.2
Mineral matter (% dw) 64.7 0.2 64.5 0.1 61.1 0.2 60.1 0.2
(mV) 34.8 0.7 58.2 0.3 36.53 0.3 57.9 0.5

FOSD, FOL TOSD, TOL FYSD, FYL TYSD, TYL

Carbon (% dw) 21.2 0.77 22 0.43 24.4 0.16 25.6 0.25

Calcium (% dw) 20.5 0.18 20 0.21 19 0.31 18 0.19
Phosphorous (% dw) 3.81 0.84 3.60 1.35 2.41 0.65 2.23 0.65
Glucose (% dry OM) 0.83 0.02 0.93 0.03 0.64 0.01 0.74 0.05
Collagen Yield (% dw) 7.9 0.3 9.6 0.2 10.6 0.2 16.9 0.3

where me is the amount of material extracted (determined by dry-
ing) from the amount of bone powder mb ; OMe , OMB and DMe , DMB ,
are the organic and dry matter fractions in the material extracted
and in the bone, respectively. Amount of mineral in collagen pow-
ders decreased as the organic matter increased as can be also
noticed from values for carbon, calcium and phosphorous (Table 1).
Taking into account that inorganic carbon represents ca. 0.95% (dw)
in 47 years old bovine cortical bone [27] values for total carbon
reported in Table 1 are indicative of the increasing amount of pro-
tein in collagen powders in the order TY > FY > TO > FO.
Organic matter in bones has been reported to be represented by
ca. 9095% type I collagen and ca. 510% non-collagenous proteins
(by weight) [4,24,26]. Results for mass spectrometry showed that
both type of proteins are present in the extracts obtained by acetic
acid (Table 2). The -1 chain of type I collagen has been identied in
all the extracts while the -2 chain has been identied only in tibia
old and femur young extracts. Also, for femur young no decorin has
been identied.
When compared to other organic and inorganic acids, acetic
acid allows higher yields of collagen in its native state (tropocol-
lagen) and which preserves natural physicochemical properties
such as spontaneous self-assembly [12,13]. As reported for the
rst time by Davidson [11], treatment of collagen with diluted
acetic acid causes an important redistribution of intermolecular
cross-linking (between adjacent tropocollagen molecules) in both
the acid-soluble and acid-insoluble collagen, and the modications
occur slowly, in the course of some days. As a result, part of the
intermolecular bonds is disrupted and tropocollagen is released
[12,13]. In this study, the effect of collagen cross-linking in bone has
been noticed not just according to the age (and as likely expected)
but also according to the anatomy (Table 1). Yield of collagen
extracted from tibia, either young or old, has been in fact signif-
icantly higher than in femur, and the increase in the yield has been
higher for young bones (Table 1). This result could be likely related
to the smaller granulometry of tibia powders with respect to femur,
even though bones have been milled under same conditions.
Two kinds of cross-links exist in collagen: the enzymatic one
(due to lysyl hydroxylases and lysyloxidase at the helical level)
which decreases with age, and the non-enzymatic one (due to gly-
cation by proteoglycans at the bril level) which increases with
age [24]. Anionic glycosaminoglycans (such as chondroitin sulphate
and dermatan sulphate in collagen type I) in small proteoglycans
(such as decorin in collagen type I) play an important role in
bridging and linking collagen brils [28]. They shape the collagen
bres by associating orthogonally to the brils through electrostatic
interactions (probably with serine residues in the triple-helical
chain) and help create the network for mineral deposition between
brils [4,29]. Small amount of proteoglycans (expressed as glucose,
Table 1) have been extracted together with collagen and minerals.
Signicantly higher values (p > 0.05) of glucose have been obtained
from old bones, and from tibia when comparing the anatomy. Even
though proteoglycans content follows total protein content, their
distribution changes with age and their post-translational modi-
cation decreases with age [24]. Presence of proteoglycans can be
also noticed from the amino acids composition of the extracted
matter reported in Table 3. Main amino acids resulted to be glycine,
proline, alanine, and hydroxyproline, the most characteristic col-
lagen residues. Higher content of leucine, tyrosine and sulphured
amino acids (methionine and cysteine) with respect to collagen
could be attributed to proteoglycans, which are rich in those
residues [30]. Higher content of other amino acids with respect
to collagen is in agreement with mass spectrometry and showed
that non-collagenous proteins are also present, as expected, since
they have been not removed before extraction. Despite to the lower
abundance (510% of total bone protein content by weight), non-
collagenous proteins are of high biologically importance; they also
contribute to collagen network stabilization and to its binding
capacity [1].
 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566 61

Table 2
Proteins identication (by LCMS/MS) in collagen solutions (FOS, TOS, FYS and TYS) hydrolysed by collagenase.

FOS TOS FYS TYS

Accession Protein MW (kDa) N of Sequence N of Sequence N of Sequence N of Sequence

peptides coverage peptides coverage peptides coverage peptides coverage
identied (%) identied (%) identied (%) identied (%)

P02453 CO1A1 BOVIN Collagen 138.9 20 5.5 21 5.9 20 7 27 10.2

alpha-1(I)
chain
GN=COL1A1
P02465 CO1A2 BOVIN Collagen 129 10 5.8 8 5.1
alpha-2(I)
chain
GN=COL1A2
P21809 PGS1 BOVIN Biglycan 41.6 4 10.3 4 10.3 5 12.2 6 16.8
OS=Bos
taurus
GN=BGN
P21793 PGS2 BOVIN Decorin 39.9 6 14.2 4 10 4 9.2
OS=Bos
taurus
GN=DCN

Table 3
Amino acids composition (%mol) of collagen powders and bovine collagen -1 chain (before post-translational modications [21]).

Amino Acid FO TO FY TY Bovine collagen -I Bovine collagen -I

chain chain
with telopeptides without telopeptides

Asp + Ans 5.4 5.3 5.7 5.4 3.2 (Asp) 3.1 (Asp)
Ans 1 1.1
Thr 2.2 2.5 2.4 2.5 1.6 1.6
Ser 3.9 4.2 4.0 4.3 3.6 3.3
Gln 2.8 2.7
Glu + Gln 9.7 9.8 9.2 9.9 4.6 (Glu) 4.6 (Glu)
Gly 24.3 22.9 22.6 23 32.8 33.5
Ala 9.4 9.2 8.4 9.2 11.6 11.9
Val 3 3.1 3.1 3.2 1.6 1.6
Ile 1.6 1.6 1.6 1.6 0.9 0.8
Leu 3.5 3.4 3.3 3.4 2.1 1.9
Tyr 1.2 1.2 1.2 1.2 0.5 0
Phe 2.2 2.3 2.1 2.3 1.2 1.2
His 2.2 2.3 2.9 2.4 0.3 0.2
Lys 2.9 3.1 2.9 3.1 3.6 3.6
Arg 3.8 3.7 3.3 3.7 4.9 5
Pro 15.2 14.7 16 14.8 22.9 (Pro + Hyp) 23.4 (Pro + Hyp)
Hyp 7 8.4 9.5 9.6
Met 1.3 1.2 0.7 1.2 0.7 0.7
Cys 1.3 1.2 1.4 0.6 0 0
Trp 0 0 0 0 0 0
Gly/(Pro + Hyp) 1.09 1 0.9 0.9 1.4 1.4
Gly/Ala 2.6 2.5 2.7 2.5 2.8 2.8

3.2. Drying methodology and collagen self-assembly
Structural properties and supramolecular architecture are
of extreme importance for the application of collagen. Drying
methodology of collagen solutions gave rise to different self-
assemblies, as likely expected [14,15,31]. Spray-drying generated
soft and quasi-spherical capsules in the diameter range 115 m
(Fig. 2, left) with a ne and uniform powder appearance at naked
eyes. The wrinkled morphology is indicative of solvent removal
even though some water and acetic acid remained trapped in the
interior (Table 1). All collagens have been able to self-assemble
under spray-drying and, apparently, particles diameter is in the
same range independently from age and anatomy. Even though
macroscopic composition of powders showed a dual phase (organic
and mineral) in each sample (Table 1), elementary analysis by SEM-
EDS revealed that content of calcium, carbon and phosphorous is
variable among particles in each sample, and this could probably
explain variability of dimension.
Micrographs relative to lyophilisation (Fig. 2, right) also showed
that all samples have been able to self-assemble whatever the age
and the anatomy. On the contrary than spray-drying, lyophilisation
generated a brous self-assembly indicating that the tropocolla-
gen molecules recovered though acetic acid are reactive and able
to cross-link each other, even though with a less degree of orien-
tation and uniformity with respect to bone matrix, at the drying
conditions used. Fibril distribution in bone presents in fact twisted
plywood packing with a periodic rotation resulting in an alter-
nating lamellar pattern [7] and a network where molecules over
40 kDa are excluded [32]. Also, the lateral structure of in-vivo col-
lagen bril can span from 20 to 400 nm in diameter arranged in
a quasi-hexagonal lattice while the length, which has been dif-
cult to study since nowadays, varies depending on the anatomical
locations (from 0.3 mm to 10 mm in rat tendon for instance, or
even end-less in other tissues as reported by some authors [4,32]).
Nonetheless, the brous self-assembly of tropocollagen recovered
from animal tissues can be modulated by several factors such as
the rate of freezing and lyophilisation, the initial concentration of
62 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566

collagen in solution, and content of proteoglycans and minerals

[33].
Different self-assemblies of collagen in the dry state gave rise
to a different stability in solution after rehydration. Values for
zeta-potential showed that collagen has a positive charge and
that rehydration after lyophilized generates more stable solutions
with respect to spray-drying. Also, tibia collagen solutions showed
greater zeta-potential, then higher stability, with respect to femur
collagen. Effect of age has been not signicant (Table 1).

3.3. Collagen powders phases

FTIR analysis conrmed a dual phase composition in bones and

in the respective collagen powders (Fig. 3ac). Mineral phase in
bones is represented by an intense bands occurring at 556 and
1012 cm1 , corresponding to the phosphate stretching [26], by a
band at 870 cm1 and bands between 1410 and 1458 cm1 cor-
responding to carbonate in type B apatite, and by bands at 1542
and 1460 cm1 corresponding to type A apatite [34]. These bands
are also visible in collagen powders. The two bands at 1647 and
1743 cm1 represent the amide I and II, respectively, while the
amide III is visible at 1240 cm1 [35]. The differences among amide
I and amide II frequencies, u = (1 -2 ) < 100 cm1 , indicates the
presence of a triple-helical conformation in bones [36]. On the
contrary, this difference has been between 120 and 130 cm1
for collagen powders indicating the loss of a triple-helical con-
formation, which is in agreement with CD spectroscopy analysis
discussed in Section 3.5. Bands at 2330 and 2360 cm1 are relative
to atmospheric CO2 [26]. The two bands at 2852 and 2922 cm1
are relative to glycosaminoglycans (linked to proteoglycans) while
band at ca. 3280 corresponding to the Fermi resonance of the C H
group [37]. The width of the band between 3100 and 3750 cm1 is to
be also attributed to the stretching of the O H bond in water which
naturally interacts with both collagen and glycosaminoglycans.
Spectroscopy in the UV/Vis range (Fig. 3d) showed a peak
at 280 nm for both collagen standard (black line) and collagen
extracted from bones. Absorption at this wavelength is due to aro-
matic amino acids tyrosine and phenylalanine which conrms the
presence of telopeptides in collagen standard. When just the helical
region is present absorption occurs in fact at 220 nm [37]. Similar-
ity in UV spectra should conrm the presence of telopeptides also
in the extracted collagen.

3.4. Thermal behaviour

Results for calorimetry show differences in the thermic

behaviour of collagen depending on age, anatomy and drying
methodology. No thermic events have been noticed by heating
samples between 10 and 50 C at 1 C/min and 10 C/min, apart
freezable water melting between 10 and 5 C (result not shown).
When heating above 50 C at 10 C/min, a unique endothermic
event between 160 and 180 C could be observed for bones (Fig. 4).
It corresponds to a one-step denaturation of the calcied collagen
due to the evaporation of strongly hydrogen-bond water responsi-
ble for the stability of the triple helix conformation with subsequent
unfolding and swelling [40,38,39]. Higher transition midpoint tem-
perature (Tm ) has been observed for old bones (Table 4); this is most
probably due to the higher degree of brils cross-linking and to the
increase with age of crystals size in the mineral fraction [24,42]. On
the contrary, higher changes in enthalpy have been observed for
young bones. Collagens bres in young bones are thinner (Fig. 1)
Fig. 3. FTIR (a,b,c) spectra. Bones (a) FO: black line, TO: red line, FY: blue line, TY:
and more exible than in old bones [24], which is reected in the
pink line. Femur old (b) bone: red line, spray-dried collagen: blue line, lyophilized
higher energy needed to be destabilised, i.e. unfolded. When com- collagen: black line. Tibia young (c) bone: red line, spray-dried collagen: black line,
paring the anatomy, either temperature or changes in enthalpy lyophilized collagen: blue line. UV spectra (d): rat tail collagen: black line, FO: purple
have been higher for tibia (Table 4). line, TO: blue line, FY: green line, TY: red line. (For interpretation of the references to
colour in this gure legend, the reader is referred to the web version of this article.)
 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566 63

Fig. 4. DSC thermograms (exo down) for bones (solid line), lyophilised collagen (dashed line) and spray-dried collagen (dashed-dotted line).

Table 4
DSC events for bones (B), lyophilised collagen (L) and spray-dried collagen (SD).

FOB TOB FYB TYB

H (J/g) 117 1.2 107 1.5 131 1.3 156 1.8

Tm ( C) 175.65 0.8 180.01 0.9 162.42 0.6 172.84 0.8

FOL TOL FYL TYL

H (J/g) 161.5 1.3 116.5 0.5 80.68 0.9 110.2 1.3
Td ( C) 200.66 0.7 243.42 0.8 177.30 1.1 179.72 1.1
H (J/g) 35.67 0.5 40.00 0.7
Td ( C) 238.26 1.2 241.39 1.5

FOSD TOSD FYSD TYSD

Tg 52.38 0.6 56.35 0.3 55.19 0.7 57.57 0.6
H (J/g) 21.97 0.9 21.27 0.4 24.13 0.6 31.67 0.9
Tc ( C) 87.11 0.4 93.64 0.6 95.91 0.7 95.33 0.7
H (J/g) 93.88 1.1 133.7 1.2 108.3 0.5 124 0.8
Td ( C) 224.55 1.3 215.84 0.8 230.88 0.9 231.22 0.7

In the case of spray-dried collagen, the event between 52 and
57 C (Tg ) relative to glass transition, corresponds to the partial
denaturation of collagen into gelatine in a still hydrated environ-
ment [39]. It has been followed by crystallisation in the temperature
range 75150 C (Fig. 4, Table 4), associated to loss of water and
acetic acid. The events between 150 and 250 C (Fig. 4) corre-
spond to a two-steps denaturation of collagen: evaporation of
hydrogen-bond water which destabilised the triple-helix rst, and
unfolding of triple-helix to a random coil conformation after. This
phenomenon has been conrmed by Bozec and Odlya [39] and
Samouillan et al. [41] which also reported that bulk degradation
of dried collagen only occurs in the range 300350 C. Spray-dried
collagen from young bones has been more heat-stable than old
bones; the contrary happened for lyophilised collagen. Old bones
lyophilised collagen in fact denatured at higher temperatures than
young bones and >200 C (Fig. 4, Table 4). Lyophilised collagen from
young bones starts denaturing by losing hydrogen-bond water at
temperatures near bones themselves (Table 4). Also, thermograms
for lyophilised powders are more similar to the bone thermograms,
which reects a brillar self-assembly in both cases.
64 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566
3.5. Secondary structure
Triple-helical, native collagen shows a characteristic CD spec-
trum with a positive peak at about 220 nm due to the electric n *
transition, and a negative minimum peak around 195197 nm due
to the magnetic * transition. Unfolding leads to a disordered
state characterised by the disappearance of the positive band at
220 nm and the presence of a negative band with reduced intensity
at about 200 nm [42,43]. In our study, result for CD spectroscopy
showed that at ambient and refrigerating temperature, collagen is
in the random coil conformation, whatever the age and the anatomy
of the bone and the drying methodology (Fig. 5a,b). This result is not
just a consequence of a thermal induced denaturation but is also to
be attributed to the thermal instability of tropocollagen at tempera-
tures in the same range of mammal tissues (28 < T < 36 C) [2,42,44].
The energetically preferred conformation of many bone and skin
proteins, in physiological solution and at the body temperature, is in
fact a random coil rather than a triple-helix [2,42,44]. This suggests
that the tropocollagen molecules are unfolded once secreted from
the cell and become folded in the triple-helical arrangement during
brillogenesis. Assembly into brils has then a stabilizing effect by
preventing further unfolding with consequent loss of the mechan-
ical properties [2,44]. Graphs of the mean polar ellipticity (mdeg)
versus wavelength for collagen solutions stored at 20 C for 5 days
(Fig. 5a,b), showed a renaturation into a triple-helix assembly,
which also conrmed the reversibility of denaturation [42,44]
Fig. 5. CD spectra for old and young collagen lyophilised and spray-dried. Solid line: 20 C, dashed line: 4 C, gray dotted line: ambient temperature.
and the reactivity of the tropocollagen recovered through acetic
acid. However, differences amongst samples have been observed.
Lyophilised tibia old collagen showed the higher degree of refolding
followed by lyophilised femur old collagen. Both showed degree of
renaturation higher than the spray-dried equivalents, and higher
than young bones collagens, either lyophilised or spray-dried, over
the 5 days of freezing. Refolding of collagen is a slow process which
takes days or weeks [42], therefore further studies would be needed
to observe renaturation over time according to the parameters
considered (temperature, pH, concentration). Finally, no isolated
-helixes have been found, being them characterised by two neg-
atives bands at 222 and 208 and by a positive band at 193 nm [44].
3.6. Principal components analysis
PCA identied three principal components to correlate the
twelve variables considered for each sample. However, stronger
correlation (>0.5) is observed only with PC1 and PC2 (Table 5).
PC1 correlates with ten variables and accounts for 67.65% of the
variance (Fig. 6). Femur old and tibia young are at the opposite
with respect to the PC1 value (Fig. 6). Higher content of mineral,
calcium and glucose is expected to be extracted from old femur
with respect to tibia young, which will be richer in protein but
will contain less GAGs, calcium and phosphorus then collagen from
old bones. Tibia old and femur young showed the highest and the
lowest PC2 value respectively, at which corresponds the highest
 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566 65

Fig. 5. (Continued)

Table 5 anatomy femur and tibia. Content of minerals and proteoglycans

Correlation between principal components and variables.
also correlated with bone age and anatomy. Despites this variability
PC1 PC2 PC3 all the collagens have been able to self-assemble under thermal
Age 0.976 0.154 0.152 treatment and to renature, however with different kinetics. The
Granulometrya 0.712 0.665 0.226 heterogeneity of type I collagen is caused mainly by differences in
OM 0.998 0.007 0.059 the quantitative conversion of lysine (Lys) to hydroxylisine (Hyl)
MM 0.999 0.020 0.037 and subsequent glycosylation of (Hyl). These differences have been
Yield 0.863 0.419 0.283
reported to be mainly tissue-specic (bones, skin, tendons, etc.),
Carbon 0.991 0.121 0.060
Calcium 0.974 0.225 0.031 which reects the characteristic biomechanical function of collagen
GAGs 0.784 0.592 0.185 in each tissue.
(SD) 0.154 0.986 0.060 Our study shows some variability of type I bone collagen pow-
(L) 0.196 0.979 0.057
ders composition depending on bone age and anatomy, as well
Td (SD) 0.840 0.510 0.188
Td (L) 0.779 0.593 0.203 as drying methodology, and this may be a crucial advantage to
a
conceive tailor-made applications in either the biological or tech-
Granulometry of bone powder.
nical sector. The possibility for a large scale treatment of discarded
and the lowest GAGs concentration, respectively, among all colla- animal tissues for the recovery of natural collagen also allows over-
gen powders. Same trends could be observed for temperature of coming difculties to synthesize tropocollagen and to generate
denaturation: collagen from old tibia has been the more heat sta- stable collagen brils. Complex intra- and extra-cellular post-
ble when lyophilized (the highest Td ) and the less heat stable when translational modications in-vivo are in fact required and are very
spray-dried (the lowest Td ). The opposite occurred for femur young expensive to reproduce through heterologous or synthetic ways.
collagen. Further studies on the effect of heterogeneity onto functionality
and bioactivity and of natural type I collagen will be carried out.
4. Conclusion
Conict of interest
Amount of collagen recovered varied signicantly according to
the bovine bone age considered 4 and 7 years, and according to the Authors declare no conict of interest.
66 V. Ferraro et al. / International Journal of Biological Macromolecules 97 (2017) 5566
Fig. 6. Relationships among variables (loading plot, red lines) and among collagen
powders (scores plot, blue dots). (For interpretation of the references to colour in
this gure legend, the reader is referred to the web version of this article.)
Acknowledgments
This publication has been written with the support of the
AgreenSkills fellowship programme which has received funding
from EUs Seventh Framework Programme under grant agreement
N FP7-26719.
Authors acknowledge the Technological Centre of Microstruc-
tures (CT) of University Claude Bernard in Lyon (France), and
the Laboratory of Magma and Vulcans (LMV) of University Cler-
mont Auvergne (France) for microstructural analysis. Nouredinne
Hafnaoui is acknowledged for amino acids analysis.
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