A Hydroxyapatite-Collagen Composite Useful To Make Bioresorbable Scaffolds For Bone Reconstruction

GIULIO D. GUERRA1,a, CATERINA CRISTALLINI1,b,

ELISABETTA ROSELLINI2,c and NICCOLETTA BARBANI2,d 1CNR Institute for Composite and Biomedical Materials, Research Unit of Pisa, Via Diotisalvi 2, I-

56122 Pisa, Italy

2Department of Chemical Engineering, Industrial Chemistry and Materials Science, University of Pisa, Via Diotisalvi 2, I-56122 Pisa, Italy

a giuliodante.guerra@diccism.unipi.it, bc.cristallini@diccism.unipi.it,

celisabetta.rosellini@diccism.unipi.it, dn.barbani@diccism.unipi.it

Keywords: Hydroxyapatite, collagen, cross-linking, scaffolds, physicochemical characterisation.

Abstract. Composites between hydroxyapatite (HA) and collagen (Col) may be used to make bio-

resorbable scaffolds for bone reconstruction. A suspension of micro-particles (average diameter Ε

30 µm) of HA annealed at 1100°C in Col solution (80:20 HA to Col weight ratio) was manufac-

tured in films by casting, and then some films were cross-linked by glutaraldehyde vapours. Cross-

linked sponges were obtained by treating the suspension with transglutaminase, and by lyophilizing

the so obtained gel. Characterization by scanning electron microscopy, water sorption test, Col re-

lease in water, thermogravimetric analysis and differential scanning calorimetry shows that the

cross-linking enhances the stability of the composite. Conversely, neither the interactions between

HA and Col, detected by spotlight FT-IR, nor the degradation by collagenase, which is a require-

ment for the bioresorbibility, are affected by the cross-linking.

Introduction

Composites between hydroxyapatite (HA) and collagen (Col) were proposed as materials useful for

bone tissue reconstruction [1,2]. To make these composites more suitable for the fabrication of bio-

resorbable scaffolds for bone tissue engineering, it may be useful to increase their toughness by

cross-linking. The present work deals with a preliminary characterisation of HA-Col films cross-

linked by exposition to pentane-1,5-dial (glutaraldehyde, GTA) and of HA-Col sponges cross-

linked with the enzyme transglutaminase (TGase).

Experimental

Materials. Stoichiometric HA was synthesized and annealed at 1100°C as previously described [3].

Micro-particles with an average diameter of about 30 µm were selected by sieving. Type I acid

soluble Col (Sigma, from calf skin, MW of about 300 KDa) was used as purchased. The HA-Col

composite was prepared by the described procedure [1]. The HA to Col weight ratio was 80:20, the

most similar to that present in the natural bone [4]. The films were prepared by casting the suspen-

sion under ventilated hood and cross-linked by exposure to GTA (Fluka) vapours, according to a

procedure already described [5]. The sponges were prepared by treating the suspension with TGase

(0,05 U/mg) in TRIS buffer (pH 7) at 37°C for 30 min, and lyophilizing the so obtained gel for ~24

hr. Also the pure Col was cross-linked by both the procedures described.

Film and sponge characterization. The composites were characterised by Scanning Electron

Microscopy (SEM), water sorption test, Col release in water, Thermogravimetric Analysis (TGA),

Differential Scanning Calorimetry (DSC), total reflection (µATR) and transmission spotlight Fou-

rier-transform infrared (FT-IR) spectroscopy and degradation by collagenase (COLase).

Advances in Science and Technology Vol. 76 (2010) pp 133-138© (2010) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/AST.76.133

All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of thepublisher: Trans Tech Publications Ltd, Switzerland, www.ttp.net. (ID: 131.114.24.252-30/09/10,16:17:02)

Results and Discussion

Type I Col, the most abundant in the human body, was used to prepare the composite. The amino

acid sequences of its three helix-shaped polypeptide chains consist mainly of glycine (Gly), in re-

peating triplets Gly-X-Y, where X and Y are mainly proline (Pro) and hydroxyproline (Hypro), re-

spectively, somewhere substituted by other amino acids, like lysine (Lys) and glutamine (Gln). The

cross-linking of Col was performed by the reactions shown in Fig. 1: Col exposure to the vapours of

a 25% GTA solution with formation of imine bridges between two Lys residues (a), and enzymatic

condensation, catalysed by TGase, of a Gln residue with a Lys residue (b).

Fig. 1. Col cross-linking by GTA (a) and by TGase (b).

These reactions do not produce the same cross-linking degree. Indeed, in the repeating (Gly-X-

Y)n amino acid sequence present in each polypeptide chain forming a Col triple helix, Lys occupies

3.6% of the X position and 9.0% of the Y position, while Gln occupies only 2.9% of the X position

and 6.9% of the Y position [6]. So, the number of Gln units is a limiting factor for the occurrence of

the reaction (b), another being the relative positions of Gln and Lys in different chains. Conse-

quently, the reaction with TGase can form less inter-chain bridges than that with GTA vapours.

134 5th FORUM ON NEW MATERIALS PART E

Fig. 2 shows the SEM micrographs of the fracture surfaces of the composites, both appearing

quite similar to cancellous bone [4].

The sponge, whose image has a lower

magnification, is more porous than the

film. The swelling percentages in water

at 37°C, shown in Table 1, are very low

and not HA-dependent for the films

cross-linked with GTA, which have

more numerous and less hydrophilic in-

ter-chain bridges. Conversely, the

sponges, having higher porosity and

lower cross-linking degree, swell much

more than the films, and the swelling

percentage of the pure Col is more than

fourfold that of the composite. These differences are likely due to the lower tightness of the struc-

ture and to the interactions of HA with Col hydrophilic sites [1,7], which prevent them from inter-

acting with water.

The release of Col in water

from the GTA cross-linked

films of both pure Col and

composite, and from the

TGase cross-linked sponges

of the composite is shown in

Fig. 3. The higher re-lease

from the sponges is likely due

to their greater porosity;

however, the very scarce re-

lease also after 90 days (4.4%

for the films and 7.1% for the

sponge) is a sign of the suit-

able stability of the cross-

linked materials in aqueous

medium.

The first derivative TGA

curves of the GTA cross-

linked films of both the pure Col (a) and the composite (b) are shown in Fig. 4. In curve (a) three

weight loss signals are visible, the weak one of the free water loss at about 50°C, the structural wa-

Fig. 2. SEM images of Col-HA 80:20 composites, cross-linked with GTA (left) and TGase (right).

Table 1. Equilibrium swelling percentages for the

films cross-linked with GTA (per cent increase of sur-

face area) and for the sponges cross-linked with TGase

(per cent increase of weight).

HA to Col ratio 0:100 80:20

Cross-linking agent Swelling percentage

GTA 3 3

TGase 1336 310

Fig. 3. Kinetics of the release of Col in water from the pure Col,

the GTA cross-linked films and from the TGase cross-linked

sponges.

Advances in Science and Technology Vol. 76 135

ter loss at about 200°C

and the Col degradation

at about 330°C. In curve

(b) the free water loss

signal appears slightly

less intense and shifted

to about 60°C, no struc-

tural water loss signal is

present, and the Col deg-

radation signal is shifted

to about 350°C and

strongly diminished in

intensity. The absence of

the structural water sig-

nal is clearly due to the

substitution of the water

molecules with the HA

crystals, observed also in

the not cross-linked

composite [1]. As for the

Col degradation signal,

the curve (b) indicates only the enhancement of the Col stability due to HA.

Table 2 shows the temperatures of Col denaturation, evaluated from the DSC thermograms of

both the films cross-linked by GTA

vapours and the sponges cross-linked

by TGase. The temperatures, much

higher than those of the not cross-

linked composites [1], confirm the

stabilizing role of the cross-linking.

Also the temperatures of the films

are higher than those of the sponges;

this fact confirms that the chemical

cross-linking is more effective than

the enzymatic one in stabilizing the

composites, because it results in the

formation of materials with tighter networks. Moreover, the higher Col denaturation temperatures of

the blends confirm the stabilizing role of HA, indicated by the Col release in water and by the TGA

measurements.

In Fig. 5 the spotlight FT-IR maps and spectra, taken in the reflection mode, of both the sponges

(a-b) and the films (c-e) are shown. In the map of a TGase cross-linked sponge (a), the red area cor-

responds to the HA granule, and the green one to the Col matrix. As the point, at which the spectra

are taken, change its position from the bulk of the matrix to the granule (b), the interactions of the

peptide —NH and —C=O groups with the PO43–

cause a splitting of the Col amide I band into a

main peak at 1658 cm–1

and a shoulder at 1639 cm–1

, as well as a shift of the amide II towards high-

er wave-numbers; as for HA, the peak at 960 cm–1

, due to the stretching of the —P—O bonds, ap-

pears shifted to 962 cm–1

. As regards the surface of a GTA cross-linked film, the chemical map (c)

shows high-middle absorbance regions, more rich in HA, and middle-low absorbance ones, more

rich in Col. The Col to HA band ratio map (d) gives more quantitative values of this distribution. In

the spectra taken at the signed points (e), a modification of the bands in that taken in the HA rich

region (blue trace), with respect to that taken in the Col rich one, can also be observed. In particular,

the amide I band appears split into a main peak at 1654 cm–1

and a shoulder at 1633 cm–1

, whereas

the amide II one is shifted from 1560 to 1567 cm–1

, and the HA peak from 960 to 965 cm–1

. These

Fig.4. First derivative TGA curves of the GTA cross-linked films of the

pure Col (a) and of the composite with 80:20 HA to Col ratio (b).

Table 2. Col denaturation temperatures, evaluated from the

DSC traces of the films cross-linked with GTA and of the

sponges cross-linked with TGase.

HA to Col ratio 0:100 80:20

Cross-linking agent Col denaturation temperature [°C]

GTA 108 170

TGase 79 121

136 5th FORUM ON NEW MATERIALS PART E

results confirm the presence, also in the composites cross-linked with both techniques, of the HA-

Col interactions observed in the not cross-linked ones [1].

The enzymatic Col degradation, shown in Fig. 6, indicates that both the cross-linking procedures

do not alter substantially the biodegradability of the protein component of the composites. At the

same time, the greater amount of degraded Col for the sponges, as well as their faster degradation

kinetics, clearly due to the

porosity of the sponges

higher than that of the films

and to the lower cross-

linking degree caused by

TGase, confirms the higher

stability of the chemically

cross-linked material.

Conclusions

All the tests made on the

cross-linked HA-Col com-

posites show that the cross-

linking increases their stabil-

ity with respect to that of the

not cross-linked ones [1,7],

so making them more suit-

Fig. 6. Degradation by COLase of pure Col, HA-Col film cross-

linked with GTA and HA-Col sponge cross-linked with TGase.

Fig. 5. Spotlight FT-IR maps and spectra, made in reflection mode. Chemical map (a) and spectra

taken at the signed points (b) of a TGase cross-linked sponge. Chemical map (c), band ratio (d) and

spectra (e) of a GTA cross-linked film.

Advances in Science and Technology Vol. 76 137

able for the fabrication of long-term bioresorbable scaffolds for bone reconstruction. As regarding

the degradation by COLase, the faster degradation of the TGase cross-linked sponges could permit

to vary the bioresorption time of the scaffolds, according to the needs of the damaged bone, simply

by varying the cross-linking technique.

References

[1] M. Gagliardi, N. Barbani, C. Cristallini, G.D. Guerra, A. Krajewski and M. Mazzocchi, in:

Proceedings of the 11th Meeting and Seminar on: Ceramics, Cells and Tissues. Annual Con-

ferences. Nanotechnology for functional repair and regenerative medicine. The role of ceram-

ics as in bulk and as coating, edited by A. Ravaglioli and A. Krajewski, pp. 182-191, CNR,

Roma (2008).

[2] G.D. Guerra, C. Cristallini, N. Barbani, E. Rosellini and M. Mazzocchi: J. Appl. Biomater.

Biomech. 7, 66 (2009)

[3] G.D. Guerra, P. Cerrai, M. Tricoli, A. Krajewski, A. Ravaglioli, M. Mazzocchi and N. Barbani:

J. Mater. Sci. Mater. Med. 17, 69 (2006)

[4] D. Felsenberg: Pharm. Unser Zeit 30, 488 (2001)

[5] C. Cristallini, G.D. Guerra, N. Barbani and F. Bianchi: J. Appl. Biomater. Biomech. 5, 184

(2007)

[6] A.V. Persikov, J.A.M. Ramshaw, A. Kirkpatrick and B. Brodsky: Biochemistry 39, 14960

(2000)

[7] G.D. Guerra, in: Proceedings of the 12th Meeting and Seminar on: Ceramics, Cells and Tis-

sues. Periodical Conferences. Surface-reactive biomaterials as scaffolds and coatings: interac-

tions with cells and tissues, edited by A. Ravaglioli and A. Krajewski, pp. 210-216, CNR,

Roma (2010).

138 5th FORUM ON NEW MATERIALS PART E