In vitro properties of a chitosan-bonded hydroxyapatite bone-filling paste

M. Ito Institute for Dental Science, Matsumoto Dental College, 1780 Gobara, Hwooka, ShlolmL Nagano 399-07, Japan

(Recewed 3 1 July 1989; revised 25 August 1989; accepted 2 November 1989)

A possible bone substitute material for dental treatment was developed and tested. The material is

composed of powdered hydroxyapatite (HA), ZnO and CaO, kneaded into a chitosan sol to make a quick-

hardening paste. A composittion was found which showed neutral pH, short setting time, and relatively

high compressive strength. The use of such a paste for the treatment of periodontal defects or the

augmentation of edentulous alveolar ridges may alleviate problems now associated with the implantation

of particulate HA, such as early migration of particles and recontouring of the implant.

Keywords: Hydroxyapatite, bone substitutes, dental materials, chitosan

The past decade has seen many favourable reports on the

use of particulate hydroxyapatite (HA) as a bone substitute in

dental treatment. Applications include repair of periodontal

defects’, ‘, implantation of tooth extraction sockets to

maintain alveolar ridge height3,4, and augmentation of

deficient alveolar ridges to improve denture support and

stability5-8. At the same time, however, dissatisfaction with

the delivery system of particulate HA has been expressed.

When placed in the implant site, the particles are loose and

can migrate beyond the intended area or be lost through

dehiscence of the incision6, ‘, ‘.

Stabilization of the implant moreover appears to

require at least one month. During this time some settling

and recontouring occurs, with a corresponding reduction in

implant sizelo,“. These problems have prompted research

on improved methods of implantation. Thus Gongloff eta/.”

report that confining the particles at the implant site with

tubes of resorbable collagen prevents particle migration;

similar success has been claimed for tubes made of

polyglycolic mesh13. Whether either of these materials will

also prevent the settling and recontouring of the implant

remains to be investigated.

A different solution to the problem of HA delivery may

lie in thedevelopment of a quick-hardening paste, containing

powdered HA, that can be easily syringed into the implant

site. This paper reports the experimental development of

such a paste, made with powdered HA (Mitsui Toatsu), and

using a sol made with chitosan (Shin Nippon Chemical) as a

binder. Chitosan was chosen because, like collagen, it is

resorbable, biocompatible, and readily available (being easily

derived from the naturally abundant macromolecule chitin);

Correspondence to Dr M. Ito.

0 1991 Butterworth-Helnemann Ltd. 0142-9612/91/010041-05

in addition, chitosan has been reported as having haemo-

static and wound-healing properties’4,‘5. Because the

chitosan sol is highly acidic, however, it was necessary to

include some substance in addition to HA to achieve

neutralization. A number of common non-toxic reagents

were tested for this purpose. The oxides of Zn (Kant0

Chemical) and Ca (Junsei Chemical), when mixed In minor

proportions with HA, were found to neutralize the chitosan

sol while inducing an acceptably short setting trme.

The purpose of the experiments described here was to

find a ratio of these three substances that would yield a

paste, when kneaded into a fixed amount of chitosan sol,

having a suitable setting time and hardness when set, aswell

as a neutral pH value. Table 7 lists the various proportions of

powdered elements tested; designations of the mixtures

hereafter follow the code given in the table. Data on the

setting times, compressive strengths and pH values of these

mixtures are reported.

The C2/26 powder m’xture appeared most promising

in that it showed a neutral pH value, one of the shortest

setting times, and the highest compressive strength of all the

mixtures tested. It was accordingly selected for further tests

of its suitability for potential application.

Samples of the hardened paste made with this mixture

were subjected to X-ray microanalysis and X-ray diffraction

pattern analysis. Experiments were also conducted which

involved several factors associated with the kneading of the

paste: the effects on pH level, setting time and compressive

strength of varying the amount of chitosan sol kneaded with

the powdered ingredients; the changes in pH level and

compressive strength over time elapsed from the onset of

kneading; and the effect on compressive strength of varying

the time that the chitosan sol is allowed to stand before

kneading.

B/omatenals 19.9 1, Vol 12 Januaw 4 1

Hydroxyepafite bone-filhng paste: M. tto

Table 1 Percentage composition of powder mixtures tested

Code CaO ZnO Hydroxyapatite

c2/Z2

C2/Z4

C2/26

C3/‘Z2

C31Z4

C3/26 C4/22

c4/24

C4/‘Z6

96 94 92

95

93

91

94 92

90

MATERIALS ARID ~ETHDDS

The sol was made by dissolving 2.5 g of chitosan SC in a solution of 2.5 g malic acid (Junsei Chemical) in 47.5 ml distilled water. Except where noted, this was allowed to stand for 24 h. The powder mixtures listed in Table 1 were kneaded manually with a spatula into the chitosan sol to make pastes. All procedures were conducted at a controlled room temperature of 23 + 1 “C.

Setting time

A 0.5 g sample of each powder mixture listed in Table 1 was kneaded into 1 .O g of chitosan sol for 30 s, and the resulting paste poured into a cylindrical mould (3 mm high, 10 mm inside diameter). The setting time was measured as the time elapsed from the onset of kneading to the point when the paste did not adhere to the end of a methyl methacrylate rod. Five assays were conducted with each powder mixture. The largest and smallest values were discarded, and the average of the remaining three values was taken as the setting time. The same procedure was followed using the C2/Z6 powder (see Tab/e I) to test the effect of increasing the ratio of chitosan sol to powder. The ratios tested used 0.5 g of powder with 1.2 g and t .5 g of sol,

pH value

For each powder mixture, five hardened specimens were made using 0.5 g each of powder and 1 .O g of sol, kneaded for 30 s and injected with a syringe into a stainless steel mould (10 mm high, 4 mm inside diameter). The moulds were covered with stainless steel plates and firmly secured with screw jacks. At 30 min after the onset of kneading, all five specimens were removed from the moulds and submerged in a bottle filled with 20 ml of Ringer’s solution. At 12 h after the onset of kneading, the pH value of the solution was measured with a pH meter (Hitachi-Horiba). Three assays were made for each powder mixture, and the average taken as the pH value for that mixture. The same procedure was followed with the CZLZ6 mixture using higher ratios of chitosan sol (0.5 g powderto 1.2 g and 1.5 g sol).

The C2/Z6 mixture was also used to test the relationship between the time elapsed from the onset of kneading and the pH value. The procedure followed the one just described, except that the specimens were removed from their moulds and submerged in the Ringer’s solution 15 min after the onset of kneading, and the pH value of the

Compressive strength

For each powder mixture, three hardened specimens were made with the procedure followed in the test of pH values. At 30 min after the onset of kneading, all three specimens were removed from the moulds and submerged in a bottle filled with 20 ml of Ringer’s solution and stored until 12 h had elapsed from the onset of kneading. The compressive strength of each specimen was measured using a universal testing machine (Shimadzu DSS-500), at a head ram speed of 5 mm/min. Three assays were conducted and their values averaged for each mixture. The same procedure was followed with the C2/Z6 mixture using higher ratios of chitosan sol (0.5 g powder to 1.2 g and 1.5 g sol).

The C2/Z6 mixture was also used for two further tests involving different times after the onset of kneading. In the first, measurement of the compressive strength was conducted at 3 h, and again at 24 h after the onset of kneading. In the second, the time elapsed between the preparation of the chitosan sol and the initiation of kneading was varied: 10, 60, 90, 120, 180 and 480 min.

~o~hological obse~ation and X-ray diffraction analysis

One gram of the C2/Z6 mixture was kneaded with 0.5 g chitosan sol, spread on a glass plate, and allowed to harden for 12 h. A 3 mm2 segment was cut from the hardened specimen, coated with vapourized gold, and the coated surface analysed with an X-ray microanalyser (Nippon Electronics JCXA-733). A larger segment, prepared in the same manner but with no coating, was examined with an X- ray diffraction analyser (Shimadzu CD-l 0), operated at 5 kV and 500 mA, using a copper Ka target and a nickel filter.

RESULTS

Setting time

The average values and 99% confidence levels for each powder mixture are presented in Figure 1. The general tendency was for a decrease in setting time with an increase in the amounts of both CaO and ZnO. The C2LZ6 mixture had a low setting time of 2 min 20 s. When the ratio of sol to the C2/‘Z6 mixture increased, however, the setting time also increased (Figure 2).

pH value

pH values of the powder mixtures ranged from a low of 6.5 (C2LZ2) toa high of 8.0 (C4/Z6). Higher pH values resulted from increases in both CaO and ZnO. Neutral values of 7.0 were obtained for two mixtures, C2/Z4 and C2/‘Z6. pH values decreased with higher ratios of chitosan sol to the C2/Z6 powder (Figure 3).

The time efapsed from the onset of kneading had moderate effect on the pH value. For the C2/‘Z6 mixture, the pH level was 6.86 f 0.2 at 15 min after kneading, and increased to a peak of 7.3 i 0.09 at 4 h. By 5 h it declined to 7.18 f 0.10, a value close to that measured at 12 h of 7.03 + 0.04.

Compressive strength

solution was measured immediately. Subsequent measure- The compressive strengths measured for the various ments were made every 5 min until 30 min after the onset of powder mixtures are presented in Figure 4. The greatest kneading, and from then on at every hour from the onset of value was for the C2LZ6 mixture (22 kg/cm2). Greater kneading until 5 h had elapsed. compressive strength was generally observed for increases

42 Biomaterials 199 1, Vol 12 January

Hydroxyapatite bone-filling paste: M. lto

6

Ia

2

20

18

-16

*; 14 w

12

; 10 .-

k8

.E 6 =: a,4 U-I

2

/l.O /l. 2 A 5

Powder/Sol Ratio cm Figure 3 pH values for different ratios of chitosan sol with the C2/Z6 mixture.

L

2 4 6 Zn 0 %

Figure 1 Setting times for the powder mixtures tested: ?WaO 2%; q CaO 3%; q CaO 4%

*

-

2 4 6 Zn 0 Oio

Figure 4 Compressive strengths for the powder mixtures tesfed. 12 hr E? CaO 2%; q CaO 3%; 0 CaO 4%

aJ 8 E i=6

n OS y2 OX5

Powder / Sol Ratio g/g *

-

Figure 2 Setting times for different ratios of chitosan sol with the C2/26 mixture r -

0.5/ l-rl- in both CaO and ZnO. When higher ratios of sol to powder

were used with the C2/Z6 mixture, the compressive

strength decreased (Figure 5). No significant variation was observed in the compres-

sive strength for different intervals between the onset of

kneading and the time of measurement for the C2LZ6

powder. Varying the time between the preparation of the

chitosan sol and the onset of kneading, however, did have a significant effect (Figure 6). Whereas values slightly under

20 kg/cm2 were observed with the C2LZ6 powder for times

0.5/ 0.5/ /l.O /1.2 /1.5

Powder/Sol Ratio s/s Figure 5 Compressive strengths fordifferent ratios ofchitosan sol with the C2/Z6 mixture.

Eiomateuals 1991, Vol 12 Januav 43

Hydroxyapatite bone-filling paste: M. ho

Figure 6

10 60 90 120 150 180 460

Elapsed Time (min)

Effect on compressive strength of varying the time elapsed between the preparation of the chitosan sol and the onset of kneading for the C2/26 mixture.

of 10 and 60 min, the compressive strength measured roughly 50% more (32.1-33.6 kg/cm*) for times of 90- 150 min. For longer intervals, values of slightly more than 20 kg/cm* were similar to that obtained under the normal procedure in which the sol is allowed to stand for 24 h before kneading.

Morphological observation and X-ray diffraction analysis

The results of an X-ray microanalysis of the hardened paste made with the C2/26 mixture are shown in Figure 7. The

even distribution of Zn suggests thorough mixture of all elements in the paste. The similarities between the distribu- tions of Ca and P indicate that the hydroxyapatite remains stable in the paste. This was further substantiated by the results of the X-ray diffraction analysis (Figure 8). The

-I ~..

Figure 8 X-ray diffraction patterns: (a) hydroxyapatite, lb) hardened C2/Z6 paste. Peaks caused by the presence of ZnO in the mixture are labelled.

Figure 7 X-ray microanalysis of the hardened C2/26 paste: (aJ composite, (b) phosphorus, (cl calcium. (d) zinc.

44 Biomaterials 199 1. Vol 12 Januaw

Hydroxyapat& hone-filltng paste: M Ito

pattern obtarned from the paste made with the C2126

mixture is virtually identical to that produced by hydroxy-

apatite alone.

DISCUSSION

The results of these tests indicate the C2/26 mixture as the

most suitable for use in a quick-hardening paste, containing

powdered HA, for possible applications as a bone substitute

material. The paste made with this mixture achieves a stable

and neutral pH value within 5 h of kneading. It also has a

short setting time (approx. 2 min), and the highest compres-

sive strength of the mixtures tested. Moreover, with a slight

increase in the ratio of chitosan sol to the powder (using

1.2 g of sol to 0.5 g powder), the setting time may be

lengthened to more than 4 min with only a moderate loss in

compressive strength, and without seriously affecting the

pH level. The loss in compressive strength may perhaps be

compensated for by letting the chitosan sol stand for at least

90 min, but not more than 150 min, before kneading the

paste, a procedure which increased compressive strength

more than 50% when a ratio of 1 .O g sol to 0.5 g powder

was used.

The paste described in this report has a slightly elastic

consistency when set. Clinical application of this paste, as a

bone substitute in dental treatment, may accordingly reduce

the incidence of inflammation in the surrounding soft tissues

noted when sharp-edged HA particles are used for this

purpose’6. Before such applications can be considered,

however, thorough evaluation of the suitability of the HA

paste as an implant material is necessary. A histological

study is currently being conducted as one aspect of this

research, and the results will be reported in the near

future.

ACKNOWLEDGEMENTS

The author wishes to acknowledge Mr S. Akahane for his

assistance with the X-ray diffraction analysis and the X-ray

microanalysis for this study. Dr W. Edwards is acknowledged

for his help with the preparation of the manuscript.

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