Biomaterials 23 (2002) 2841–2847

Study on the stabilisation of collagen with vegetable tanninsin the presence of acrylic polymer

B. Madhan, C. Muralidharan*, R. Jayakumar

Central Leather Research Institute, Council of Scientific and Industrial Research, Adyar, Chennai 600 020, India

Received 30 May 2001; accepted 2 December 2001

Abstract

Collagen, a unique connective tissue protein finds extensive application as biocompatible biomaterial in wound healing, as drug

carriers, cosmetics, etc. A study has been undertaken to stabilise Type-I collagen of rat-tail tendon using plant polyphenol (Acacia

Mollissima) in the presence of an acrylic polymer. It has been found that collagen fibres pre-treated with acrylic polymer followed by

the treatment with Acacia Mollissima exhibited an increase in hydrothermal stability by 251C. Infrared spectroscopic studies display

the changes in the spectral characteristics of native and treated collagen films. Transmission electron microscopic and circular

dichroic studies provide an insight into the understanding of the improved stabilisation of collagen, due to treatment with acrylic

polymer and plant polyphenols. The study is expected to enhance the biomaterial applications of collagen tissues. r 2002 Elsevier

Science Ltd. All rights reserved.

Keywords: Vegetable tannins; Polyphenols; Acrylics; Collagen; Stabilisation; Biomaterial

1. Introduction

Collagen is the major structural component ofconnective tissues. It is an important biomaterial findingseveral applications as prosthesis, artificial tissue, drugcarrier and cosmetics. Vegetable tannins are polyphe-nolic compounds present in the plant extracts. These arecompounds with molecular weights in the range of500–3000Da [1,2]. The chromatographic studies indi-cate that vegetable tannin extracts are heterogeneouspolyphenolic species [3–5]. In the process of leathermaking, vegetable tannins are used in the stabilisation ofskin [6]. Skin predominantly contains collagen, theabundant protein in animals. There are 19 differenttypes of collagen [7], of which the type I collagen is themain component of skin, tendon, bone and other tissues.This protein is present as chains wound in tight triplehelical form [8], which are organised into fibrils of greatstrength and stability [9]. The structure of collagen isstabilised by inter- and intra-chain hydrogen bonds [10]and by water-mediated hydrogen bonds [11,12]. Some ofthe oligomers of chromium are known to interact with

collagen [13–15] bringing about irreversible matrixchanges and long-range ordering, thus imparting higherhydrothermal stability [16].

Polyphenols in the form of vegetable tannin extractsare being used in the process of stabilisation of skintracing back to the history of mankind. Polyphenolspresent in extracts of green tea are known to possessanti-carcinogenic activity. Extracts of green tea mostlycontain derivatives of catechins [17,18]. Pharmacologi-cal activity of tannins has been investigated, and theirantitumor–antiviral effects have been revealed earlier[19]. Polyphenols of vegetable tannin extracts arecapable of cross-linking with collagen through theformation of multiple hydrogen bonds [20,21]. In ourearlier work, we were able to obtain leather of higherhydrothermal stability by the treatment with vegetabletannins in the presence of acrylic polymer [22]. Collagenis the major leather-making protein [23]. Hence, it wasfelt necessary to study the stabilisation of collagenbrought about by the usage of vegetable tannins in thepresence of an acrylic polymer. An attempt has beenmade to understand the process of stabilisation ofcollagen. Rat-tail tendon (RTT) which predominantlycontains type I collagen has been used in this study.Wattle or Mimosa (Accasia Millissima) has been chosenas the vegetable tannin source for the present study.

*Corresponding author. Tel.: +91-44-491-5730; fax: +91-44-491-

1589.

E-mail address: cmurali62@yahoo.com (C. Muralidharan).

0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 4 1 0 - 0

Mimosa belongs to a group of condensed tannins, whichbasically contains molecules with flavanoid structure[24–26].

2. Materials and methods

2.1. Materials

Mimosa tannin extract from Tanzanian source wasused as a source of polyphenol. Commercial acrylicpolymer with an average molecular weight of 8000 wasemployed for the treatment of collagen along with thepolyphenol.

2.2. Structural elucidation of acrylic polymer

The commercial acrylic polymer chosen for the studywas characterised using FT-IR, C13 NMR and H1 NMRspectroscopic analysis.

2.3. Treatment of RTT fibres

Collagen fibres teased from the tails of 6-weeks oldSprague-Dawley rats were used for the study. Teasedcollagen fibres were washed with 0.9% NaCl at 41C, toremove the adhering soluble proteins. The RTT waswashed extensively in double-distilled water at 41C.Three different experiments were carried out with thesefibres. One set of the tendons was treated with 1%acrylic polymer for 12 h and another set was treated with5% vegetable tannins (Acacia Mollissima) for 12 h.Another experiment was carried out by treating thetendons initially with 1% acrylic polymer for 1 h,followed by treatment with 5% vegetable tannins for12 h. Acrylic polymer and vegetable tannins used for thetreatment were weakly acidic.

2.4. Shrinkage temperature determination

The shrinkage temperature of RTT fibres treated withacrylics and vegetable tannins were measured using amicro-shrinkage meter as described below using thestandard method [27]. A small strip of fibre was cut andplaced on a water-grooved microscope slide. The slide inturn was placed on a heating stage along with amicroscope mounted above the heating stage. The rateof heating was maintained at 21C/min. The temperatureat which the fibre shrinks to one-third of its length wastaken as the shrinkage temperature.

2.5. Interaction studies with collagen film

The collagen solution prepared from RTT [28] wascast on a glass and airdried in a laminar flow hood. Thecollagen films were treated with acrylic, vegetable

tannins as mentioned in Section 2.3. The treatedcollagen films were washed with water air-dried andcharacterised using FT-IR.

2.6. Circular dichroic (CD) spectroscopic studies

Collagen solution extracted from RTT by a knownprocedure [28] was used for CD studies. The collagencontent of the solution was estimated by standardprocedure [29]. Conformation of native collagen in 5mm

acetic acid was recorded in the far UV region usingJasco 715 CD spectropolarimeter. The collagen solution(0.3mg/ml) was treated with acrylic polymer (3mg/ml),and vegetable tannins (3mg/ml), and the reactionmixture was investigated for the conformationalchanges.

2.7. Electron microscopic studies

The native and the treated RTT samples wereexamined under a transmission electron microscope(JEOL-1200EX) to identify the topological distributionof vegetable tannins by analysing the change in bandingpatterns of the native collagen.

3. Results

3.1. Characterisation of acrylic polymer

The FT-IR spectrum of the acrylic polymer is shownin Fig. 1a. The carbonyl (C¼O) stretching vibration at1708 cm�1 is due to the carboxylic group; following thissignal, there is a shoulder signal at 1635 cm�1 and thismay be due to the carboxyl group of carboxylate anion(Fig. 1a). A series of signals noticed in the range of1479–1178 cm�1 support the structure of poly-methacrylic acid. The confirmation of the sample beingpolymethacrylic acid is established using H1 and C13

NMR spectroscopy shown in Fig. 1b and c. The H1

NMR shows four signals in the range of 1.07–2.07 ppm,which are due to a-methyl and methylene proton of thebackbone. The C13 NMR shows a group of signals inthe range of 183–185 ppm, which are due to carbonylcarbons of the carboxylic group and its anion. This isfurther supported by the signals observed at 55–57, 47,48 and 19–20 ppm, respectively. The presence of a signalat 19–20 ppm clearly indicates the presence of a-methylgroup thereby establishing the polymethacrylic acidstructure. Additional signals as observed in the spectrumcould possibly be due to other additives present in thecommercial polymer. No attempt has been made toelucidate these signals, as they were not expected toinfluence any major change in the collagen matrix.

B. Madhan et al. / Biomaterials 23 (2002) 2841–28472842

3.2. Hydrothermal stability of collagen fibres

The shrinkage temperatures measured for the nativeand treated RTT fibres are shown in Table 1. Theshrinkage temperature for native collagen fibre has been

found to be 581C. The RTT fibres treated with theacrylic polymer and vegetable tannin extract exhibitshrinkage temperatures of 681C and 761C, respectively.The RTT fibre treated with acrylic followed by vegetabletannin treatment exhibits a hydrothermal shrinkagetemperature of 861C.

3.3. Interaction of collagen with acrylic polymer and

vegetable tannins

The IR spectra of the untreated collagen film andfilms treated with acrylic polymer, vegetable tannins anda combination of acrylic with vegetable tannins areshown in Fig. 2a–d, respectively. The major feature ofthe IR spectrum of collagen film (Fig. 2a) is the amide Iband between 1640 and 1660 cm�1, which arises fromthe stretching vibration of C¼O groups of amidegroups in protein. The intense absorption between 1500and 1600 cm�1 is due to the amide II mode, observed at1550 cm�1 in the spectrum for collagen (Fig. 2a), whicharises from N–H stretching vibration strongly coupledto the C–N stretching vibration of collagen amidegroups. Collagen has high proportion of glycine andproline, which makes it unique compared to otherproteins. These two amino acids might be responsiblefor some of the spectral characteristics between 1200and 1400 cm�1. Signals in the spectral region of 1200–1400 cm�1 absorption are generally attributed to theamide III, arising due to the C–N stretching and N–H inplane bending from amide linkages. The absorption seenat 1238 cm�1 is attributed to amide III vibration. Inaddition, significant absorption due to CH2 waggingvibrations from the glycine backbone and proline sidechains are also seen in this region. The absorption seenat 1340 cm�1 is attributed to CH2 wagging vibration ofthe proline side chain. The C–N stretching vibration ofthe cyclic proline may also contribute absorptionbetween 1200 and 1350 cm�1.

The vibrations due to amide I, II and III correspondto peaks at 1654, 1550, 1238 cm�1, respectively. All thethree characteristic peaks are seen in the case of all otherspectrums (Fig. 2b–d), where the collagen film has beensubjected to treatment. The characteristic acrylic,carbonyl stretching vibration at 1708 cm�1 is also seenin the case of the collagen film treated with acrylicpolymer (Fig. 2b), but the peak is not sharp since it is

Fig. 1. (a) FT-IR spectrum of the acrylic polymer; (b) H1 NMR

spectrum of the acrylic polymer; and (c) C13 NMR spectrum of the

acrylic polymer.

Table 1

Shrinkage temperature (1C) of native and treated collagen fibres

RTT collagen fibres Shrinkage temperature (1C)

Native 58

Acrylic polymer 68

Vegetable tannins 76

Acrylic+vegetable tannins 86

B. Madhan et al. / Biomaterials 23 (2002) 2841–2847 2843

coupled with the peak of amide I vibration, whereas thischaracteristic acrylic peak is missing in the case of thecollagen spectrum treated with acrylic and vegetabletannins (Fig. 2d). This is an interesting observation,which shows a possibility of the carboxyl group of theacrylic polymer being changed by the influence ofvegetable tannins. The peaks in the region of 1000–1350 cm�1, which are seen in the case of the collagen filmtreated with vegetable tannins (Fig. 2c and d), are due tothe bending of aromatic groups of the vegetable tannins.In the untreated collagen film, stretching of the N–H ofthe amino acids is observed at 3324 cm�1, whereas in thecase of the collagen film treated with vegetable tannins,the peak is shifted towards the higher frequency rangearound 3350 cm�1 and broadened. This stretchingcannot be due to free hydroxyl stretching, because thehydroxyl stretching vibrations appear only above3550 cm�1. Therefore, this characteristic peak shouldbe possibly because of the hydrogen bonding of the

vegetable tannins with the collagen. In the case ofcollagen film treated with a combination of acrylic andvegetable tannins, the broadening is higher than that inthe earlier case.

Transmission electron photomicrographs of RTTcollagen treated with vegetable tannins, acrylic polymerand acrylic followed by vegetable tannins are presentedin Fig. 3. The photomicrograph of RTT treated withvegetable tannins (Fig. 3a) does not show a clearbanding pattern. This could probably be due to coatingof the fibres with tannins. In the case of the photo-micrograph of RTT treated with acrylic polymer alone(Fig. 3b) and vegetable tannins in the presence of acrylicpolymer (Fig. 3c), clear banding patterns are observed.In the photomicrograph of RTT treated with acrylicpolymer, a higher order of fibre splitting is observed asreported earlier [30]. Addition of vegetable tannins tothe acrylic-polymer-treated RTT also brings about acertain degree of orderliness in the fibre packing. This issupported by a higher shrinkage temperature observedin the case of RTT treated with acrylic polymer followedby vegetable tannins, where the shrinkage temperature isfound to increase from 681C (RTT treated with acrylicpolymer) to 861C. The observed increase in shrinkagetemperature can be explained through long-rangeordering and cross-linking of fibres [31] treated withacrylic polymer and vegetable tannins.

The CD spectra of native and treated collagensolutions are shown in Fig. 4. CD spectrum of nativecollagen is dominated by the p2p� amide transition at196.5 nm and a positive n2p� transition peak at 220 nm.The CD spectrum of the collagen treated with acrylicshows an increase in molar ellipticity at 196.5 nm, whichindicates that there is a moderate unfolding of collagenon the introduction of acrylic polymer. When vegetabletannins are added to the collagen solution after treatingwith acrylic polymer, a change in amplitude of thespectrum, which orients towards the spectrum of nativecollagen, is observed.

4. Discussions

Vegetable tannins, as a result of their general chemicalcharacteristics, impart hydrothermal stability to col-lagen fibrils predominantly by interaction with basicgroups like lysine and argentine [32], which are locatedat the ‘band’ region in the collagen structure [33]. Theband structure of collagen contains sterically bulkyacidic and basic amino acid side chains, which are openand accessible for interactions with vegetable tannins.Thus, the reaction of vegetable tannins with the collagenbrings about orderliness, which necessitates higher heatenergy for the denaturation to take place.

Polymers may increase the shrinkage temperature oftendon by increasing the volume fraction of collagen

Fig. 2. FT-IR spectrum of the collagen film: (a) Native collagen film;

(b) collagen film treated with acrylic polymer; (c) collagen film treated

with vegetable tannins; and (d) collagen film treated with acrylic

polymer followed by vegetable tannins.

B. Madhan et al. / Biomaterials 23 (2002) 2841–28472844

within the fibrils [34,35]. It therefore appears thatvegetable tannins in the presence of acrylic polymerincrease the shrinkage temperature by shifting theequilibrium between native and denatured forms ofcollagen towards a more compact native form by stericexclusions. A similar proposal has been made to explainthe increased melting temperature of DNA in thepresence of polymer [36]. The acrylic polymer used forthe treatment of collagen fibre was predominantlypolymethacrylicacid, which is likely to open up thecollagen molecule through electrostatic forces [37], asthey contain active sites for charge interactions. Such anopening up is likely to facilitate vegetable tanninmolecules to diffuse into the collagen matrix and bringabout enhanced ordering, which is responsible forimproved hydrothermal stability of collagen, as seen inthe case of RTT collagen fibre treated with acrylicfollowed by the treatment with vegetable tannins. Aschematic representation of the interaction of collagenwith acrylic followed by vegetable tannins is mentionedin Fig. 5. The reaction scheme mentioned in the figure isa representation of the interactions taking place at theinter-fibrillar level in the collagen tendons. The acrylicsbehave as negatively charged molecules under theapplied conditions and hence can interact with positivelycharged regions of the collagen. The collagen matrixopens up because of such interaction. On the addition ofvegetable tannins, compactness of the collagen matrixincreases due to the interaction of vegetable tannins withcollagen. Vegetable tannins used in the study containpolyphenolics such as catechin, gallic acid, etc. as mainconstituents. The hydroxyls are the main functionalgroups of interaction for these tannins. These hydroxylgroups can form a hydrogen bond with the side chain

Fig. 3. Transmission electron photomicrographs of RTT collagen

fibres: (a) collagen fibre treated with vegetable tannins; (b) collagen

fibre treated with acrylic polymer; and (c) collagen fibre treated with

acrylic polymer followed by vegetable tannins.

Fig. 4. CD spectrum of collagen solutions: (FF) native collagen

solution (0.3mg/ml); (FF) collagen solution treated with acrylic

polymer; (- - - - - ) collagen solution treated with vegetable tannins; and

(– - –) collagen solution treated with acrylic polymer followed by

vegetable tannins.

B. Madhan et al. / Biomaterials 23 (2002) 2841–2847 2845

groups of polar amino acids like lysine, arginine,aspartic acid and glutamic acid. The other amino acidslike serine and threonine can also involve in thehydrogen bond formation with the polyphenolic mole-cules [38]. All these amino acid residues can act ashydrogen bond donors and acceptors. Since thesepolyphenolics have several hydroxyl and carboxylgroups, they can form hydrogen bonds at multiplepoints, imparting additional stability to the fibre matrix.

Acknowledgements

The authors wish to thank Dr. T. Ramasami,Director, CLRI for his interest and encouragement.The authors also thank Dr. T. Narasimhaswamy andDr. A. Rajaram, scientists, CLRI for their help in

carrying the presented work. Thanks are also due to themembers of the Chemical Laboratory of CLRI for theirsupport.

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