In vitro and in vivo degradation of films of chitin and its deacetylated derivatives

Biomoteriols 16 (1997) 567-575 0 1997 Elsevier Science Limited Printed in Great Britain. All rights reserved PI1

SO142-9612

(96)

0142.9612/97/$17.00

00167-6

In vitro and in tivo degradation of films of chitin and its deacetylated derivatives Kenji Tomihata and Yoshito Ikada Research Center for Biomedical Japan Chitin

was deacetylated

chitins.

The specimens

100 mol% solution

(chitosan). casting

mol%),

lowered Unlike

The equilibrated

observed

crystallinity their

without

degradation lysozyme

films

properties,

a maximum

at 37”C, while

were

deacetylated

towards

highly

cationic

primary

amines

Keywords:

Chitin,

Received 1 December

(chitosan). since

as their

the films

compared

of these

in buffered

deacetylated

showed

derivatives

in the molecule. deacetylated

c

including

aqueous

1995; accepted 22 September

Science

chitosan,

became solution

was very

less rapidly

higher.

The in vitro

implanting

the films

high for chitin chitin.

The films the tissue

mild, although

All rights tissue

in

and

Interestingly,

was very

to the

polymers.

of pH 7 containing

deacetylated

biodegradation,

and the minimum

may be ascribed

films occurred

Limited.

the tensile

320

are crystalline

biodegradation.

chitosan

while

content

by subcutaneously

slower

1997 Elsevier

chitins,

water

and chitosan

with that for the 73.3 mol%

deacetylated

(chitosan),

by the

73.8 (68.8

232 (73.3 mol%),

of deacetylation

was studied

specimens

52.4 (chitin),

0 and 68.8 mol%

that the rate of in vivo biodegradation

chitin,

from these

The maximum

both chitin

deacetylated

68.8, 73.3, 84.0, 90.1 and

and 49.7 wt%

between

degree

and thoroughly

were

197 (68.8 mol%),

deacetylated

the in vivo degradation

Biodegradable materials are becoming increasingly more important in the biomaterial field, particularly for wound healing, tissue reconstruction and controlled drug delivery. The main advantage of biodegradable over non-biodegradable materials is the disappearance of implanted foreign materials from the body as a result of their biodegradation. This is a marked contrast with non-biodegradable biomaterials which might elicit foreign-body reactions from the host’s defence system during their long-term contact with a living structure. For medical applications, we need many kinds of biodegradable materials that possess a variety of physico-chemical properties as well as biodegradation kinetics. In some instances a hard material with a high initial mechanical strength and a low degradation rate is required, while some surgical applications need soft materials with a high degradation rate. Most synthetic biodegradable materials such as polyglycolide and polylactides have high strengths, while natural products like collagen are low in mechanical strength but exhibit excellent cell adhesion. Representative natural biodegradable polysaccharCorrespondence

prepared

of the films

57.8 (90.1 mol%) 244 (chitin),

out by immersing

partially

were

contents

partially

Shogoin, Sakyo-ku, Kyoto 606,

by 0 (chitin),

in vitro and in vivo degradations

more than 73.3 mol%

reaction

of 150pm

of chitin,

or minimum,

the back of rats. It was found which

deacetylated

water

and 4339 mm-’

for a specimen

was carried

68.8 mol%

were

by deacetylation

physical

passing

with NaOH to obtain were

61.8 (84.0 mol%),

293 (90.1 mol%)

strength

extents

Films with a thickness

method.

of the water-swollen

(84.0 mol%), tensile

to various

Kyoto University, 53 Kawahara-cho,

used in this study

64.2 (73.3 mol%),

strengths

Engineering,

they had

reserved

reaction,

lysozyme

1996

ides include starch, mucopolysaccharides and chitin. The latter and the completely deacetylated product, chitosan, are very attractive biomaterials, because both of them are crystallizable’ and have a high potentiality to provide materials of excellent mechanical properties” 3. The chemical structures of their repeating units are shown below:

Lo iH,

Chitosan

Chitin

Although chitin and its deacetylated derivatives seem to have very promising properties as biomaterials4-lo, few papers have been published on the biodegradation of chitin and its deacetylated derivativesgS’1-‘7. The

to Dr Y. Ikada. 567

Biomaterials

1997.

Vol.

18 No. 7

568

Degradation

of chitin

purpose of this work is to investigate the physical and biological properties of chitin and its partially and thoroughly deacetylated derivatives. Chitosan, the 100% deacetylated chitin, is known to be present in the animal world, similar to chitin.

MATERIALS

AND

METHODS

Materials Chitin powder and chitosan powders having various degrees of deacetylation were supplied by Katokichi Bio Co. Ltd. (Kagawa, Japan). The partially deacetylated chitins were obtained by hydrolysis with saturated aqueous NaOH solution at GO-70°C for about 5 h’“. The degrees of deacetylation of chitin powders were 68.8, 73.3, 84.0, 90.1 and 100 mol% and hereafter we will call the deacetylated derivatives C-69, C-74, c-84, C-90 and C-100, respectively.

Preparation

of films of chitin

Films of chitin were prepared according to the method of Tokura et al.‘. Briefly, the powders were suspended in formic acid at room temperature, followed by freezing at -20°C for 24 h. When this freeze-thaw cycle was repeated several times, the turbid gel turned gradually to a clear gel. The gel was dispersed in formic acid by the addition of a small amount of dichloroacetic acid. The polymer films were prepared by casting their solutions on a glass plate, followed by drying at room temperature. The thickness of the dried films was 150 pm on average.

Preparation of films of partially and thoroughly deacetylated derivatives The films of partially and thoroughly deacetylated derivatives were prepared according to the method of Mochizuki et al.lg, by casting 1 wt% aqueous polymer solution containing 1 wt% acetic acid on a Petri dish and drying at room temperature. The resulting film was placed in 3 wt% NaOH solution, containing 50wt% ethanol solution to neutralize the acid, and washed several times with distilled water. The thickness of the dried films was approximately 150 pm.

Measurement of physico-chemical

properties

The water content of films of chitin and its deacetylated derivatives was determined as follows. The films were immersed in O.lM phosphate-buffered saline (PBS; pH 7.4) for 2Oh at 37°C to equilibrium, removed from the PBS, and then placed between two dry pieces of filter paper to remove excess solution. The wet films were weighed and then placed in a vacuum oven for 6 h at 60°C under 1 kPa. The dried films were weighed and the water content was calculated by the following equation: Water content

(wt%) = [(wet film weight

- dry film weight)/wet

film weight]

x 100

The static water contact angle of the water-swollen films was measured at 25°C with the conventional sessile drop method. The zeta potential of the films was determined at pH 6.8 and a KC1 ionic strength of 1.0 x 10e3 using a cell unit”. Biomaterials

1997. Vol. 18 No. 7

and its deacetylated

derivatives:

K. Tomihata and Y. lkada

Mechanical testing of films Mechanical measurement was conducted for the wet films after swelling in double-distilled water (DDW) at 25°C for 20h. Prior to the mechanical testing, the film thickness was measured using a film thickness gauge. The films were subjected to tensile testing using an Autograph AGS-5D (Shimadzu Inc., Kyoto, Japan] at a film length of 25 mm and a rate of 40% strain per minute (cross-head speed = 10 mm mini’). The tensile strength was calculated as the breaking load divided by the initial film cross-sectional area. In vitro

degradation

Three-times recrystallized egg-white lysozyme was purchased from Sigma Chemical Co. (USA) and used without further purification. The films of chitin and its deacetylated derivatives of a known weight were lysozyme solution in 0.1 M immersed in 4 mgmll’ PBS at pH 7.4 and 37C. After determined intervals of time the films were taken out from the lysozyme solution, rinsed with DDW, dried and weighed. The extent of in vitro degradation was expressed as the percentage of the weight of the dried film after lysozyme treatment.

In vivo degradation A pouch was made in the subdermal tissue of the back of 25 Wistar rats. The films of chitin and its deacetylated derivatives of a known weight were sterilized with ethylene oxide gas prior to implantation, soaked in sterile PBS (pH 7.4) and inserted into the pouch. The pouch and the skin incision were closed with a single silk suture. After determined intervals of time the rats were killed and the samples were retrieved to weigh after drying at 60°C for 6 h under 1 kPa. The retrieved samples were rinsed with a copious amount of DDW and dried to a constant weight, which was recorded. The extent of in vivo degradation was expressed as percentage of the weight of dried films after implantation, similar to the degradation with lysozyme.

Histological observation After determined intervals of time the rats with the implanted films were killed and all of the films were explanted without loss from the rats together with the surrounding tissue. Following fixation with 10 wt% formalin and enbedding in paraffin they were sectioned with a microtome and stained with haematoxylin-eosin (HE). Some film fragmentation occurred during their sectioning. Twenty-five rats were used for this histological study and each of them was implanted with one piece of film.

RESULTS Hydration The equilibrated water content of the films of and deacetylated derivatives is shown in Figure water swelling was performed using PBS at the thoroughly deacetylated Interestingly, (chitosan, C-100) has a water content of 50 wt%,

chitin 1. The

37°C. chitin which

Degradation

of chitin

and its deacetylated

derivatives:

K. Tomihata

and Y. lkada

Table 1

Interfacial

C-O (chitin) C-69 c-73 C-84 c-90 C-100 (chitosan)

40 -//0

100

60

60

Degree of deacetylation (mol%) Figure 1 The water content

of films of chitin and its deacetylated derivatives swollen with phosphate-buffered saline at 37°C as a function of the degree of deacetylation.

569 properties

of deacetylated

chitin films

0 (degree)

Zeta potential

69.5 57.6 56.7 49.0 48.6 49.8

0.52 0.12 0.66 0.61 1.06 0.28

(mV)

This implies that the mechanical properties of these films are not directly related to their hydration extent. It seems probable that the strength as well as the elongation of films may be greatly influenced by the overall crystallinity and microcrystalline size of these films on which deacetylation will have a large effect.

Surface properties is almost the same as that of chitin before deacetylation. Deacetylation of chitin converts the acetamide group into a primary amine group, which is generally more hydrophilic than its acetamide. Therefore, deacetylation may first lead to an increased water content of the films, as shown in Figure 1. However, the film water content no longer increases but decreases with the degree of deacetylation, and the C-100 film, chitosan, shows the lowest water content among the deacetylated derivatives. This indicates that hydration of the deacetylated derivatives is also governed by factors other than the content of the acetylated hydrophobic group in the molecules.

Since the surface properties of biomaterials are influential on the tissue response when implanted, the water contact angle and the zeta potential of the films of chitin and its deacetylated derivatives were measured. Table I gives the results. As expected, the film surface becomes less hydrophobic upon deacetylation of chitin to chitosan. It is interesting to note that all the films have practically zero or slightly positive zeta potentials in the neutral medium, although the deacetylated derivatives are regarded as cationic polymers carrying primary amine group?. When cellulose film was cationized by the Mickel reaction, the zeta potential did not become positive”.

Mechanical properties

Degradation

The films of deacetylated derivatives underwent hydration to greater extents than that of chitin when swollen with water at 37°C to equilibrium, unless it was completely hydrolysed. It is interesting to compare the mechanical properties of chitin with those of deacetylated derivatives in the hydrated state. The results are shown in Figure 2. As can be seen, the deacetylated derivatives have higher elongation at break than chitin before deacetylation. On the other hand, the mechanical strength of chitin swollen with PBS is lower than that of the deacetylated derivatives if the degree of deacetylation is higher than 80mol%.

In general, polysaccharides are degraded by enzymatic hydrolysis. Chitin and chitosan are also reported to follow this principle”-*7~23-25. Indeed, they exhibit no appreciable degradation when brought into contact with aqueous neutral media containing no enzymes at room temperature. In vitro hydrolysis Chitin readily undergoes lysozyme14.16’23. The

degradation in vitro

in PBS containing degradation of

80 g

60

400

60 60

5 5

300

F g cn

200

'40 20 lOOL//

' 60

0

1 60

I

100"

20

Time (hr)

Degree of deacetylation (%)mOl% Figure 2 Mechanical

properties of films of chitin and deacetylated derivatives swollen with phosphate-buffered saline at 25°C as a function of the degree of deacetylation: 0, strength; 0, elongation.

,

30

Figure 3 In vitro degradation deacetylated derivatives pH 7.4 at 37°C: 0, chitin; A, C-100 (chitosan).

of films of chitin and its in 4 mg ml-’ lysozyme solution of 0, C-69; 0, C-73; W, C-84; A, C-90;

Biomaterials

1997, Vol.18 No. 7

Degradation

570

of chitin

derivatives:

K. Tomihata

and Y. lkada

deacetylated derivatives with 4 mg ml- 1 lysozyme is shown in Figure 3. In contrast to chitin, deacetylated derivatives with degrees of deacetylation higher than 73 mol% are virtually resistant against the enzymatic hydrolysis at this lysozyme concentrationz5. On the other hand, the 69 mol% (C-69) deacetylated derivative undergoes intermediate biodegradation between chitin and chitosan.

Q @ F ._ c ._

and its deacetylated

80

!I L E .P

In vivo hydrolysis The biodegradation results of films of chitin and its deacetylated derivatives implanted in the subcutaneous tissue of rat back are shown in Figure 4. It is seen that chitin and C-69 are degraded quite rapidly compared with other deacetylated derivatives. A remarkable finding is a significant difference in the biodegradation between C-69 and C-73. The difference in the degree of deacetylation between the two films is only 4mol%. This is more clearly seen in Figure 5, where the initial slopes of the curves in Figure 4 are plotted as the initial resorption rate against the degree of deacetylation. It is unclear to us which kind of enzyme is responsible for the in vivo degradation of chitin and its slightly deacetylated derivative (C-69). One of the most effective catalysts for the hydrolysis may be lysozyme, which is known to be ubiquitous in the body. On the contrary, it seems very likely that rats have no hydrolytic enzyme that can catalyse the scission of chemical bonds in chitosan (C-100). In other words, chitosan is practically not biodegradable, in marked contrast to chitin.

80

z

40. 0

2

4

8

8

10

12

14

Time (Week) Figure 4 In vivo degradation of films of chitin and its deacetylated derivatives when subcutaneously implanted in rats: 0, chitin; 0, C-69; 0, C-73; n , C-84; A, C-90; A, C100 (chitosan).

Tissue reaction

-..J==._

60

60

100

Degree of deacetylation (mol%) Figure 5 Dependence of the initial resorption chitin and its deacetylated derivatives on deacetylation.

rate of films the degree

of of

Tissue responses to chitin and its derivatives were studied by implanting the films in the subcutaneous tissue of rats for different periods of time. The subcutaneous tissue, where the films were implanted, was taken out together with the films and stained with HE after formalin fixation for the histopathological study. The microphotographs taken after implantation for 1, 2, 4, 8 and 12 weeks are given in Figures 6-10 respectively. Figures 8-l 0 do not include any results for chitin and C-69, because implantation of these films for longer than 4 weeks resulted in almost complete resorption into the body.

Chitin Figure 6 Optical (haematoxylin-eosin Biomaterials

1997.

micrographs staining, Vol. 18 No. 7

of the original

C-69 subcutaneous magnification

tissue x40).

around

films

of chitin

and

C-69

implanted

in rats

for

1 week

Degradation

of chitin

Figure 7 Optical rats for 2 weeks

and its deacetylated

derivatives:

K. Tomihata and Y. lkada

571

Chitin

C-69

c-73

C-84

micrographs of the subcutaneous tissue around films of chitin (haematoxylin-eosin staining, original magnification x40).

As is seen in Figure 6, a relatively severe inflammatory reaction was observed in the tissue surrounding the chitin and C-69 films implanted for 1 week. However, this tissue response subsided 2 weeks after implantation, as shown in Figure 7. After 4 weeks of implantation one observe the continuing can

and its deacetylated

derivatives

implanted

in

inflammation, but the extent was very low, as seen in Figure 8. When films were implanted for 7 weeks, encapsulation started around the implanted films by a collagenous tissue. This is shown in Figure 9. Figure 10 reveals that implanted films were more strongly encapsulated 12 weeks after implantation, Biomaterials

1997, Vol. 18 No. 7

Degradation

572

Figure 8 Optical micrographs for 4 weeks (haematoxylin-eosin

similar to conventional, ials.

of the subcutaneous staining, original

non-biodegradable

of chitin

tissue around films magnification x40).

biomater-

DISCUSSION As demonstrated above, deacetylation of the acetamide groups of chitin yields products with primary amine groups which may exhibit physical and biological properties different horn the starting chitin. First, the hydrophobicity of chitin decreases upon deacetylation, but the hydration extent does not increase in proportion to the degree of deacetylation, as shown in Figure 3. Interestingly, the film of chitosan (C-100) with the highest degree of deacetylation has the lowest water content among the deacetylated derivatives. This apparently peculiar result can be explained in terms of the microstructure of the deacetylated derivatives, most probably the overall crystallinity. As is well known in polymer chemistry, partial derivatization of crystalline polymer chains introduces defects in the crystalline structure. As both chitin and chitosan are crystalline polymers, the crystallizability of chitin and chitosan must be reduced by deacetylation and Biomaterials 1997, Vol. 18 No. 7

and its deacetylated

of deacetylated

derivatives:

derivatives

K. Tomihata and Y. lkada

of chitin

implanted

in rats

acetylation, respectively, resulting in an increase in the water content of the films when swollen with PBS. This result is quite similar to that of other crystalline polymers. For instance, poly(viny1 alcohol) is also insoluble in water at room temperature, but becomes water-soluble when partially acetylated, because of reduction in the crystallinityz6. The mechanical strength of crystalline polymers will also become poor if the crystallinity is partially lost, as demonstrated in Figure 2. The high mechanical strength of the waterswollen deacetylated films must be due to the microcrystallites present in the films, which will also prevent dissolution in water of pH 7. In contrast to the hydration, the biodegradation of the films of chitin and its deacetylated derivatives seems not to be governed by the physical microstructure but by the chemical structure of the films. As shown in Figures 3 and 4, lysozyme and other enzymes present in rat cannot catalyse hydrolysis of chitosan, which is free of acetamide groups. These enzymes are able to hydrolyse only chitin and its deacetylated derivatives still possessing a certain amount of the acetamide group in the molecules23-25. It is not necessary for the polysaccharide molecules to carry the acetamide group

Degradation

of chitin

and its deacetylated

Figure 9 Optical micrographs for 8 weeks (haematoxylin-eosin

derivatives:

of the subcutaneous staining, original

K. Tomihata

and Y. lkada

tissue around films magnification x40).

on each of the repeating units, because, as shown in Figure 6, C-69 exhibits a degradation rate almost similar to chitin, although 69mol% of the acetamide group of chitin has been deacetylated to the amine group. However, 69mol% of deacetylation is the limit for rapid biodegradation, since the C-73 film is very slowly degraded. Probably, the frequency of certain sequential chitin units, in other words, the length of the chitin chain block, may be crucial for the molecular recognition by the hydrolytic enzymes, as pointed out by other workers14B23’24. As is obvious from Figures 6-10, the tissue response to the implanted chitin and its deacetylated derivatives is very mild except for the chitin and C-69 films, which have been most rapidly degraded. This is in agreement with the well-known fact that rapidly biodegradable biomaterials elicit an acute inflammation reaction due to a significantly large production of low-molecular-weight compounds within a short time. Figure 6 shows that numerous granulocytes are present around the implanted films, probably to phagocytose the degradation products. If biodegradation of implanted materials takes place very slowly, the tissue response would be mild, similar to non-biodegradable biomaterials which do not leach any

of deacetylated

573

derivatives

of chitin

implanted

in rats

compounds recognizable by phagocytotic cells. In such a case, fibrous encapsulation of the implanted films will occur, as is observed in Figures 9 and 10. A remarkable finding in the present study is a very mild tissue reaction towards the completely deacetylated chitin (chitosan). It has been pointed out that cationic groups on a material surface invoke irreversibly strong cell adhesionz7 and hence a severely inflammatory reaction when such cationic materials are implantedz8. However, the film of chitosan did not elicit any significant foreign-body reaction, although each of the repeating units of the chitosan molecule has a cationic primary amine. This apparent contradiction may be explained in terms of the zeta potential of the film of chitosan. As shown in Table 1, the zeta potential of this 100% deacetylated chitin is not highly positive, but very close to zero. This implies that the surface of the film of chitosan is so bioinert that it may not give any strong stimulus to the surrounding tissue2g-31. This almost zero zeta potential of the film of chitosan is probably due to the weak basicity of the primary amine and the preferential adsorption of anions on the film surface. The surfaces of conventional polymers such as polyethylene, silicone and polytetraBiomaterials

1997. Vol. 18 No. 7

Degradation

Figure 10 Optical micrographs for 12 weeks (haematoxylin-eosin

of the subcutaneous staining, original

of chitin

tissue around magnification

fluoroethylene have zeta potentials of several negative tens of millivolts. In summary, it may be concluded that chitin is a biodegradable polymer, in contrast to chitosan. The biodegradation rate of chitin can be controlled to some extent by the variation in the deacetylation. Deacetylation of chitin enhances the mechanical strength and reduces the inflammatory tissue reaction. This milder tissue response to chitosan than chitin, although the chitosan molecule carries primary amines, may be explained by the practically zero zeta potential of the film of chitosan.

and its deacetylated

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Biomaterials 1997,Vol. 18 No. 7