Dimethyl suberimidate cross-linked pericardium tissue: Raman

spectroscopic and atomic force microscopy investigations

Maria Jastrzebskaa,*, Justyna Zalewska-Rejdaka, Roman Wrzalikb, Antoni Kocotb,Bogdan Barwinskic, Iwona Mrozc, Beata Cwalinaa

aDepartment of Biophysics, Faculty of Pharmacy, Medical University of Silesia, Ostrogorska 30, 41-200 Sosnowiec, PolandbDepartment of Biophysics and Molecular Physics, Institute of Physics, University of Silesia, Universytecka 4, 40-007 Katowice, Poland

cInstitute of Experimental Physics, University of Wrocław, Plac Maxa Borna 9, 50-204 Wrocław, Poland

Received 7 September 2004; revised 2 November 2004; accepted 2 November 2004

Available online 18 January 2005

Abstract

Chemically stabilized pericardium tissue is widely used as a tissue-derived biomaterial for the preparation of bioprostheses such as heart

valves or vascular grafts. The bifunctional imidoester dimethyl suberimidate (DMS) belongs to the wide class of the cross-linking reagents

and is often used to cross-link a variety of proteins, including collagen matrices and collagen-based tissues. Raman spectroscopy in the wide

frequency range 200–4000 cmK1 and contact mode atomic force microscopy (AFM) have been employed to investigate the structural

changes and chemical bonds in DMS cross-linked porcine pericardium tissue. It has been found, that in addition to the commonly accepted

reaction with the 3-amine groups of lysine or hydroxylysine residues, DMS may interact also with the carbonyl CO and amide NH groups of

the peptide bond in collagen. Our paper presents for the first time spectral evidence for the peptide contribution to the formation of DMS–

collagen cross-links. The results confirm also possible competition between the hydrolysis of the free imidoester group and cross-linking

reaction. Products of the partial alkaline hydrolysis of DMS have been found in the spectra. The observed changes in the surface topography

of the fibrils as well as in their spatial organization in the tissue support the formation of both intra- and interfibrillar cross-links in DMS-

stabilized tissue.

q 2005 Elsevier B.V. All rights reserved.

PACS: 87.14.-g; 87.64.Je; 07.79.Lh

Keywords: Cross-linking; Collagen; Raman spectroscopy; Atomic force microscopy

1. Introduction

Chemically stabilized pericardium tissue is widely used as

bioprostheses in replacement surgery of the vascular system,

e.g. in arterial reconstruction or cardiac valve replacement

[1]. Chemical stabilization with different reagents, e.g.

glutaraldehyde (GA) or dimethyl suberimidate (DMS),

improves the mechanical properties, reduces immuno-

genicity and enzymatic or chemical degradation processes

of the material. However, there are several factors limiting

the functional lifetime of the tissue-based bioprostheses such

0022-2860/$ - see front matter q 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.molstruc.2004.11.040

* Corresponding author. Tel.: C48 322925541.

E-mail address: maja@slam.katowice.pl (M. Jastrzebska).

as calcification, cytotoxicity of the cross-linking reagent or

mechanical fatigue of the material [2].

Pericardium tissue is composed mainly of collagen type I

[3]. Chemical stabilization introduces cross-links into

molecular structure of collagen fibrils. Multiple cross-

linking techniques have been explored in an attempt to find

the ideal procedure to stabilize the collagen-based tissue

[1,2].

The bifunctional imidoester DMS has been employed to

cross-link a variety of proteins and supramolecular

structures, including collagen matrices and collagenous

tissues. Imidoesters are highly specific reagents for amine

groups in proteins, as shown in [4,5]. DMS is widely used as

an alternative fixative reagent to GA because of its lower

cytotoxicity. For example, rat fibrous dermal collagen has

Journal of Molecular Structure 744–747 (2005) 789–795

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M. Jastrzebska et al. / Journal of Molecular Structure 744–747 (2005) 789–795790

been cross-linked with DMS for use as dermal implant,

since DMS was found to be less cytotoxic than glutar-

aldehyde. DMS has also been evaluated as a cross-linking

agent for human dermis and porcine aortas for use as

bioprostheses [6].

Mechanisms of the DMS–protein interactions are not

fully understood. It is known, that DMS reacts with the amine

groups of collagen to form diamidine derivative structures

[6]. Another study [6,7] explains, that diimidoesters like

DMS do not polymerize and form cross-links only with free

amine groups from lysine or hydroxylysine residues that are

separated by a distance equivalent to the molecular length of

DMS, i.e. about 11 A. This may restrict the number of cross-

links being introduced into the collagen matrix. DMS cross-

linked collagenous membranes seem to be more biocompa-

tible than glutaraldehyde-treated membranes as revealed by

subcutaneous implantation studies [6]. DMS cross-linked

membranes promote fibroblast migration and proliferation

and act as a scaffold for tissue regeneration.

Raman spectroscopy is a very powerful tool for studying

the structure and molecular interactions of many complex

molecules. This method was successfully used for studying

the mechanism of GA-collagen cross-linking [8]. One of the

greatest advantages of this technique is its ability to provide

information about the structure and interactions of biomo-

lecules in their microenvironment within intact cells and

tissue. The technique is not destructive and does not require

homogenization, extraction or the use of dyes, labels or

other contrast-enhancing agents. As a vibrational technique,

Raman spectroscopy is more advantageous than IR

spectroscopy, e.g. the structural properties of molecules in

aqueous solution are more easily observed [8].

Atomic force microscopy (AFM) is being widely applied

to study surface topography of many biological structures,

at high resolution, in liquid or gaseous environments, so that

materials can be studied in hydrated conditions close to their

native state [9].

The aim of the present work was to study structural

changes and chemical bonds, which are involved in DMS

cross-linking of pericardium tissue, using Raman spec-

troscopy and atomic force microscopy.

2. Experimental

2.1. Materials

Pericardium tissues from hearts of 5–6-month-old pigs

were obtained fresh from slaughter. Fatty tissues and

sections with heavy vasculature or attached ligaments

together with a serosal layer were gently removed from

each pericardium. Three 15!10 mm rectangles were

excised from each pericardium.

2.2. Chemical treatment of pericardium tissue

Stabilization with DMS was performed using 1% DMS

(SIGMA) solution in 0.2 M Tris buffer (pH 9) at 4 8C for 3 h

in dark. After chemical treatment pericardium tissue was

rinsed three times with distilled water and then kept in

phosphate buffer (pH 9) at 4 8C until the measurement could

be made.

2.3. Deuteration

Samples of the pericardium tissue designed for deutera-

tion were transferred into D2O (deuterium oxide 99.8%,

SIGMA). Deuteration was performed in excess of D2O,

5 ml/mg of dry tissue, for 0.5 h.

2.4. Raman spectra

Raman spectra were recorded using a LabRam Raman

spectrometer (Jobin-Yvon-Horiba) comprising an Olympus

BX40 confocal microscope. It was equipped with a grating

monochromator and a charge-coupled device (CCD)

Peltier-cooled detector (1024!256 pixels). The incident

laser excitation was provided by an air-cooled argon laser

source operating at 514.5 nm. The power at the exit of a

50! objective was 50 mW. The spectra were recorded with

resolution of 2.5 cmK1, a collection time of 60 s and an

accumulation of 10 scans. In order to avoid undesirable

Rayleigh scattering, two notch-filters were used cutting the

laser line at 100 cmK1. Pure silica was used to calibrate the

spectrometer.

Baselines of the individual Raman spectra were adjusted

with 20–30 point baseline correction functions using

GRAMS 32.

Spectral intensity was normalized using the CC

vibrational mode of phenylalanine (Phe) aromatic ring

(1004 cmK1) as an internal standard.

Native and DMS cross-linked pericardium tissues are

materials of considerable surface heterogeneity. Raman

spectra were recorded from 10 different points of each

sample surface. The results presented in the paper show

representative records of the Raman spectra.

The measurements were conducted at room temperature

(20 8C) without thermostating.

2.5. Atomic force microscopy

AFM images were obtained in the contact mode, using

the NanoScope E, Digital Instruments, USA, equipped with

the OTR8 probe (Veeco NanoProbee). The length and the

spring constant of the applied V-shaped cantilever were

200 mm and 0.15 N/m, respectively. The constant forces

used were about 10 nN. All measurements were performed

in air, at room temperature. The samples were placed onto

clean surfaces of glass microscope slides. After 20 min,

M. Jastrzebska et al. / Journal of Molecular Structure 744–747 (2005) 789–795 791

when the excess of water had evaporated from the sample

surface, the AFM images were recorded.

Two standard AFM signals, i.e. the height and the

differential signal deflection were registered. The height

image corresponds to the topography of the sample, whereas

the deflection image is sometimes more useful in direct

observations. The lateral and height resolutions were 10 and

1 nm, respectively. All AFM images were processed using

the software package WSxM (Spain).

3. Results and discussion

3.1. Raman spectra

Figs. 1 and 2 show the Raman spectra of porcine

pericardium tissue in its native state and after DMS

treatment. The spectra were recorded in two wavelength

ranges, 200–2000 and 2000–4000 cmK1, respectively.

Frequencies and assignments of the bands are collected in

Table 1.

The spectrum in the range 2000–4000 cmK1 has been

recorded for the deuterated tissues. H to D exchange shifts

the OH vibrational mode of bulk water to lower frequencies.

After short equilibration time in D2O (e.g. 0.5 h), the

remaining broad signal in the range 3200–3700 cmK1

Fig. 1. Raman spectra of the native and DMS-stabilized porcine pericardium tiss

Table 1. Peaks, which are discussed in the paper in terms of the DMS–collagen in

inset. Band at 1667 cmK1 is assigned to the CaN stretching vibration [28].

corresponds to the more tightly bound water with the slow

rate of H to D exchange. Following literature data, the

distinct peak at 3328 cmK1 is assigned as an NH vibration

from amides or amines, as shown in Table 1. Because of the

very slow rate (over several days) of the amide- or amine-

proton exchange in collagen, the reduction in the NH

vibrational mode seems to be negligible [10].

Collagen type I is the main component of the fibrous layer

of pericardium tissue. According to the earlier findings [11],

the spectra in Figs. 1 and 2 show bands, which are

characteristic for collagen type I. The basic collagen

molecule consists of a right-handed superhelix formed by

three helical strands. The positions of the amide I

(1655–1667 cmK1) and amide III (1241–1272 cmK1)

bands as well as a strong C–C stretch band around

939 cmK1, support the presence of the helical conformation

in a collagen molecule [12,15]. The observed increase in the

band intensity around 940 cmK1 can point to a stabilizing

effect of the DMS cross-linking on collagen helical structure.

The interactions of the DMS with the collagen from

porcine pericardium tissue result in formation of stable

cross-links. This chemical modification induces changes in

the Raman spectra of the tissue. The analysis of the position

and the intensity of the Raman bands for native and DMS-

stabilized tissues allowed us to recognize the following

types of the DMS–collagen interactions:

ue, in the frequency range 200–2000 cmK1. Peak assignments are given in

teractions are in bold. Raman spectrum of the solid DMS is presented as an

Fig. 2. Raman spectra of the deuterated native and DMS-stabilized porcine pericardium tissue, in the frequency range 2000–4000 cmK1. The inset shows the

repeated measurements in the frequency range 3200–3700 cmK1.

M. Jastrzebska et al. / Journal of Molecular Structure 744–747 (2005) 789–795792

3.1.1. Interaction with amine R–NH2 group

According to literature data [6,7], the principal reaction

involves formation of cross-links between the methoxy

group of DMS and 3-amine group of lysine or hydroxylysine

residues in collagen with the release of methanol, as seen in

Scheme 1. Fig. 2 shows Raman spectra for deuterated

tissues with the distinctly seen peak at 3328 cmK1, which is

assigned to the NH vibration from the amines or amides

(Table 1). For more clear presentation, the inset in Fig. 2

shows the repeated measurement in the frequency range

3200–3700 cmK1. Both figures do not show any changes in

the 3328 cmK1 peak after DMS treatment. This result can be

explained by the reaction presented in Scheme 1. According

to the scheme, each molecule of DMS introduces two NH2

groups, simultaneously two amine groups from lysine or

hydroxylysine in collagen are involved in cross-linking

reaction. As a result, there is no change in the amount of

the NH2 groups after DMS treatment and in consequence,

no change in peak intensity at 3328 cmK1.

3.1.2. Interaction of peptide NH group with DMS

The amide III band originates from the NH in plane

deformation around 1270 cmK1 coupled to the CN stretch-

ing mode at 1247 cmK1. As shown in Fig. 1, after DMS

treatment an increase in the CN band intensity and,

simultaneously, a decrease in the NH band intensity are

observed. This effect can be explained by the formation of a

secondary amide type bond between the NH group of

collagen and methoxy OCH3 group from DMS with the

release of methanol, as seen in Scheme 2.

Release of methanol presented in Scheme 2 can be sup-

ported by the increase in the peak intensity at 1319 cmK1,

which is assigned as the OH vibration (Table 1).

3.1.3. Interaction of peptide CO group with DMS

Fig. 1 shows a decrease in the amide I band intensity at

1665 cmK1 for the DMS cross-linked tissue. The main

contribution to the amide I band is the peptide carbonyl

stretching vibration in the range 1655–1667 cmK1. The

observed decrease in the band intensity can be interpreted as

the formation of covalent bond between carbon of the

peptide bond and NH group of the DMS molecule. The

reaction is shown below (Scheme 3).

The observed decrease in band intensity of the carbonyl

bond should be accompanied by the increase in the CaN

bond vibration, according to Scheme 3. In the

Raman spectrum of the solid DMS, the band around

1667 cmK1 is assigned to the CaN stretching vibration

Table 1

Band assignments of Raman spectra of porcine pericardium tissue in native state and after DMS cross-linking treatment

Native pericardium DMS-pericardium Assignment of vibrational mode Ref.

722 719 (CH2) deformation in phase [33]

760 758 (CCO) [34]

772 (PO) stretch [13]

814 814 (COC) deformation [8,33,34]

856 855 (CC) aromatic (Pro, Tyr) [13–15]

875 874 (CC) aromatic (Hyp, Try) [14,15,35]

922 922 (CH2) wagging out of phase [15,34]

939 939 (CC) stretch [8,12,13,36]

965 (PO) sym. stretch [37]

1004 1003 Phe, (CC) ring breathing [13,15,33,38]

1032 1034 (CC) stretch of pyridine ring [20,33]

1065 1065 (COC) deformation [21,22]

1083 1083 (CN)/(CC) deformation [15,23,33]

1102 1099 (COC) stretch asym. [24]

1124 1124 (COC)/ (CN) stretch [15,25,26]

1164 1166 (CC) out of plane/phase [33,34]

1178 1177 (CC) stretch, Tyr [14,15,33–35]

1206 1206 (C–C6H5) stretch [13,15,33,38]

1247 1245 (CN) stretch/Amide III [12,15,26,27,34]

1273 1270 (NH) deformation/Amide III [12,15,26,27,34]

1319 1322 (C–OH) bend [24]

1343 1340 (CH3, CH2) deformation [15,23,28,34]

1397 1395 (CH2) bend [33,34]

1428 1427 (COOK) stretch [15]

1452 1452 (CH) deformation/scissor [26–30,34]

1573 1573 (NH2) scissor/Amide II [8,24]

1665 1664 (CaO) stretch/Amide I [12,26,31,32]

2200–2800 (OD) stretch [10,11]

2800–3000 (CH2, CH3) stretch sym., asym. [30,32–34]

3067 3067 (CaCH) stretch [33,34,38]

3328 3326 (NH) stretch sym. [8,10,30]

sym., symmetric; asym., asymmetric.

Scheme 3.

Scheme 2.

Scheme 1.

M. Jastrzebska et al. / Journal of Molecular Structure 744–747 (2005) 789–795 793

Fig. 3. AFM height (A) and deflection (B) images (1.3!1.3 mm) for native

pericardium tissue. The curve (E) shows the axial profile along the line 1 for

the native collagen fibril. AFM height (C) and deflection (D) images (0.7!

0.7 mm) for the DMS-stabilized pericardium tissue. The curve (F) shows the

axial profile along the line 2 for the DMS cross-linked collagen fibril.

M. Jastrzebska et al. / Journal of Molecular Structure 744–747 (2005) 789–795794

(inset to the Fig. 1). Conjugation of the CaN with amide

carbon lowers the CaN stretching frequency to a value

below 1667 cmK1 located on the shoulder of the amide I

band. As a result, an increase in the CaN vibration is very

hardly seen on the shoulder of the strong amide I band.

As it was mentioned earlier, the increase in the OH peak

intensity at 1319 cmK1 can also correspond to the formation

of the hydroxyl group, which is attached to the amide

carbon.

The analysis of the Raman spectra shown in Fig. 1

reveals also a spectral evidence for the possible partial

hydrolysis of the DMS. The evidence presented in the

literature points to two major hydrolytic pathways for an

imidoester, giving either oxygen ester and ammonia or

amide and alcohol [4]. Yuthavong et al. [4] found, that at

least 84% of DMS (10 mM) was allowed to hydrolyze in

0.1 M triethanolamine buffer, pH 7.0, with the release of

ammonia. At higher pH values, e.g. pH 9, reaction of DMS

and lysine or hydroxylysine is improved and the yield of

cross-linking products is raising. Under our experimental

conditions, it has been found, that slow alkaline hydrolysis

of DMS can occur. Fig. 1 shows, that after DMS treatment

there is an increase in the 1428 cmK1 peak intensity, which

is assigned to the COOH vibration. At alkaline pH, the

oxygen ester is converted to the suberic acid with the release

of alcohol. The observed increase in the band intensity at

1555–1575 cmK1 for the DMS treated tissue can support

the presence of ammonium ions. The bands visible in the

spectral region 1555–1575 cmK1 are usually assigned to the

amide II band, which is weak or not seen at all in Raman

spectra. The band in the region 1555–1575 cmK1 can also

correspond to the scissoring vibration of the amine NH

group (Table 1).

3.2. AFM images

Fig. 3 shows the height and deflection images for native

(Fig. 3A,B) and DMS-stabilized (Fig. 3C,D) porcine

pericardium tissue. The axial profiles taken from the height

images along the marked lines are also presented in the

figure (Fig. 3E,F). They describe the surface topography of

collagen fibrils.

For the native pericardium tissue, collagen fibrils ran

mostly parallel to each other. A regular transverse

D-banding pattern with a period of about 67 nm, consisting

of high ridges and shallow grooves is distinctly seen

(Fig. 3E). This result is in good agreement with that

previously obtained from the AFM studies for native

collagen type I fibrils [16–18]. The native D-banding

pattern results from the quarter-staggered arrangement of

collagen molecules within the fibril. However, no common

opinion regarding the staggered molecular structure have

been yet presented in the literature. Some recent models

assume not molecular stagger but a staggering of micro-

fibrils made of six or seven non-staggered parallel-aligned

collagen molecules [19].

For the DMS stabilized tissue the D-pattern is signifi-

cantly disturbed (Fig. 3F). As seen in the axial profile, there

are numerous periods that are longer than 70 nm. Moreover,

the spatial organization of fibrils within the tissue is

disturbed, i.e. fibrils are tangled and running in different

directions. The observed considerable changes in the

surface topography of collagen fibrils can confirm formation

of cross-links between collagen molecules within the fibril.

Changes in the spatial organization of fibrils within the

tissue are most probably due to the formation of the inter-

fibrilar cross-links.

4. Conclusions

The analysis of the Raman spectra in the wide frequency

range 200–4000 cmK1, for the native and DMS-stabilized

porcine pericardium tissue, allowed us to recognize different

types of the DMS–collagen interactions. It has been found,

that in addition to the commonly accepted reaction with the

3-amine groups of lysine or hydroxylysine residues, DMS

M. Jastrzebska et al. / Journal of Molecular Structure 744–747 (2005) 789–795 795

may interact also with the carbonyl CO and amide NH

groups of the peptide bond in collagen. Our paper presents

for the first time the spectral evidence for the peptide

contribution to the formation of DMS–collagen cross-links.

The results confirm also possible competition between

the hydrolysis of the free imidoester group and cross-linking

reaction. Products of the partial alkaline hydrolysis of the

DMS molecules have been found in the spectra.

The observed changes in the surface topography of the

fibrils as well as in their spatial organization in the tissue

support the formation of both intra- and interfibrilar cross-

links in DMS-stabilized tissue.

Acknowledgements

Support of the Committee for Scientific Research (KBN)

grant no. NN-5-156/04 is gratefully acknowledged. One of

the authors (IM) thanks Institute of Experimental Physics,

University of Wrocław, for financial support (grant no.

2016/W/IFD/03).

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