Regulated release of drug microspheres from inflammation responsive degradable matrices of crosslinked hyaluronic acid

Journal of Controlled Release, 25 (1993) 133-143 0 1993 Elsevier Science Publishers B.V. All rights reserved

133 0168-3659/93/$06.00

COREL 0084 1

Regulated release of drug microspheres from inflammation responsive degradable matrices of crosslinked hyaluronic acid Nobuhiko

Yui, Jun Nihira,

Teruo

Okano

and Yasuhisa

Sakurai

Institute of Biomedical Engineering, Tokyo Women’s Medical College, Shinjuku, Tokyo, Japan (Received 8 September

1992; accepted in revised form 2 1 December 1992)

Heterogeneous structured devices consisting of lipid microspheres (LM) in degradable matrices of hyaluronic acid (HA) crosslinked with polyglycerol polyglycidylether were prepared and their inflammation responsive degradation was investigated in vivo. Hydroxyl radicals produced by the reaction of H202 and FeSO, induced a rapid but limited degradation of the crosslinked HA gels. Zero ordered release of LM was achieved in proportion to the degradation of crosslinked HA gels according to surface-controlled mechanism. In vivo implantation experiments revealed the quantitative degradation of the HA gel in response to inflammation. Therefore, crosslinked HA gels may be promising devices as inflammation (stimulus)-responsive degradable matrices for implantable drug delivery. Key words: Crosslinked hyaluronic acid; Lipid microsphere; ture; Inflammation responsive drug delivery

Introduction Regulated drug release from biodegradable polymer matrices has been widely examined in order to release dispersed or dissolved drug in proportion to degradation of the polymer matrix [ 11. The degradation of aliphatic polyesters such as poly (lactic acid) and poly (glycolic acid) occurs uniformly throughout the polymer matrix, which causes both burst effects and inactivation of incorporated drug. Thus, drug release from such biodegradable polymers is difficult to predict, and the development of novel drug formulations using surface degradable polymers has been desired. Correspondence to: Nobuhiko Yui, present address: Japan Advanced Institute of Science and Technology, 15 Asahidai, Tatsunokuchi, Ishikawa 923-12, Japan.

Hydroxyl

radical; Heterogeneous

struc-

Recently, several types of biodegradable polymers have been extensively studied to obtain drug release which is responsive to a biological and external stimulus [2,3]. Polymers such as poly (ortho esters), polyacetals and polyanhydrides have been used to effect drug release in proportion to the erosion rate at the polymer surface. Surface erosion (degradation) of these polymers takes place through hydrolysis of labile linkages in hydrophobic polymer chains [ 2 1. Stimuli-responsive polymer erosion may be achieved by the incorporation of pH-sensitive labile linkages into the polymer chains. Enzymesubstrate reactions, which produce a local pH change may then be used to vary the erosion rate of the polymers. However, it seems quite difficult to regulate the release rate of drugs via pHsensitive polymer erosion. In order to achieve an auto feed-back drug delivery system using biodegradable polymers, these polymers require that

134

degradation only occurs via a specific internal signal with a sufficient life time to ensure the desired degradation and appropriate delivery of drugs. Hyaluronic acid (HA) is a linear mucopolysaccharide composed of repeating disaccharide subunits of N-acetyl-D-glucosamine and D&Icuronic acid. HA has been extensively used in vivo as a therapeutic agent for ophthalmic surgery and arthritis [ 4-61. In the living body, HA is known to be degraded by two mechanisms: (1) via hyaluronidase as a specific enzyme [ 5,7] and (2) via hydroxyl radicals as a source of active oxygen [8-l 5 1, The degradation of HA by hydroxyl radicals may be dominant and rapid as compared to that by hyaluronidase if HA is injected in the proximity of inflammatory reactions. In the first stage of the inflammation process, an enhanced capillary permeability permits the accumulation of polymorphonuclear leukocytes and other phagocytic cells at the inflammation site. These phagocytic cells, which may be activated by immune complexes and other inflammation-generating compounds, produce hydroxyl radicals, that serve as a bactericidal agent. Since the viscosity of synovial fluid and HA solutions after exposure to enzymatically generated superoxide was reduced in vitro, several reports have emphasized the depolymerization of HA by hydroxyl radicals [ lo- 15 1. In our previous paper, the degradation of crosslinked HA gels was evaluated in vitro by both hydroxyl radicals and hyaluronidase, and in vivo by inflammation [ 161. One of the specific features of these gels was that the degradation profiles of the HA gels followed the kinetics of surface-controlled polymer degradation, which may be promising for designing a new biodegradable hydrogel. One question could arise through this issue: how can any drug be incorporated in such a highly water swollen hydrogel in order to perform a degradation-controlled release of that drug? When any drug is introduced in a biodegradable hydrogel, chemical immobilization of the drug on the hydrogel matrix has been generally discussed [ 171. The objective of such drug-

immobilized polymer systems, a so called ‘polymer-drug conjugate’, is to provide a prolonged action of the drug by the hydrolysis or biological scission of the covalent bonds. This design is based on the general assumption that drug released from polymer chains by the scission of covalent bond is active. However, it is considered that drug covalently bonded to macromolecular chains could have a possibility of the drug inactivation prior to the drug release. Furthermore, drug immobilization into polymer chain could be limited in terms of the amount of immobilized drug, which might be influenced by the solubility of drug itself. From this perspective, a heterogeneous device constructed of a degradable HA gel matrix containing drug microreservoirs (lipid microspheres) was proposed as a new designed implantable device for drug delivery. This heterogeneous structure was found to enable degradation-controlled release of lipid microspheres. Furthermore, inflammation responsive degradation of these implanted devices in rats was more quantitatively evaluated by forming carrageenin-induced granulomas close to the implanted site. The heterogeneous device is feasible for regulated drug release from biodegradable hydrogels and for protected inactivation of drug with the hydrophobic nature of microreservoir.

Materials and Methods Preparation of crosslinked HA gel-containing lipid microspheres Hyaluronic acid (HA) was supplied from Shiseido Co., Tokyo, Japan, as a dry powder. The molecular weight of HA was determined to be approximately 8.7 x 1O5by an intrinsic viscosity measurement [ 18 1. Polyglycerol polyglycidylether (PGPGE) were supplied from Nagage Chemical Industry Co., Tatsuno, Japan. A suspension of lipid microspheres (LM) with an average diameter of 0.2 - 0.4 ,um was purchased from Otsuka Pharm. Co., Tokyo, Japan, as a 20 wt.% aqueous suspension (Intralipid@, Kabi Vitrum AB, Stockholm, Sweden). Bovine testicu-

135

.

CH,\,CH-CH,-O-(CH2-CH-CH,-O-CH-CH,-O),-CH,-CH*-CH,-,CH* 0

0

OH 7 CH2-CH,GcH2 0

(PGPGE)

lar hyaluronidase (HAase) (825 units/mg HA solid) and carrageenin type IV (p-carrageenin) from Gigartina aciculaire and G. postillata were purchased from Sigma Chemical Co. St. Louis, U. S. A. The chemical structures of HA and PGPGE are shown above. Crosslinked HA gels were prepared as follows [ 161: 20 wt.% HA solution in 1 N NaOH (4.0 ml), which contained 1.O g of HA, was prepared and degassed well. 20 wt.% LM suspension ( 1.O ml) was mixed with HA solution, followed by the addition of PGPGE ( 1.54 g) to the HA solution. The molar ratio of PGPGE to the repeating unit of HA was 1.O. The mixture was vigorously mixed and was injected to a glass cell (inner diameter: 1 cm, height: 3 mm) in order to react at 60’ C for 15 min. The gel obtained was immediately placed into a large excess of distilled water/ethanol ( 1/ 1 vol), and was neutralized by the addition of hydrochloric acid. The gels were then washed with distilled water/ethanol several times. Ethanol in the gel was replaced with distilled water proir to additional experiments. The weight of both equilibrated gel in water and its freezedried one were measured for calculating the water

content. The gel was opaque and its water content was found to be 99.5% in distilled water. After freeze drying, the morphology of drying crosslinked HA gels was observed with a scanning electron microscope (JEOL, Tokyo, Japan, JSM-5300LV). In vitro degradation of crosslinked HA gel The degradation of crosslinked HA gels by hydroxyl radicals generated by H202 and FeSO, was examined in vitro [ 161. Unless otherwise noted, the procedure was as follows. A slab (20 x 20 x 2 mm) of crosslinked HA gel was immersed in 10 mM FeSO, solution ( 100 ml) for 2 days in order to form HA-Fe2+ complex. Then, the degradation of crosslinked HA gel was initiated by immersing the gel into a solution of H202 (50 or 100 ml) at different concentrations with stirring. The concentration of H202 was determined spectrophotometrically by using the reaction of o-dianisidine at 500 nm [ 191. The degradation of HA gel was estimated by measuring the residual weight. The system solution (1.0 ml) was sampled, and LM release from the crosslinked

136

HA gel was estimated by measuring the transmittance of the sampling solution at 500 nm by using spectroscopy (Japan Spectroscopy Co. Tokyo, Japan, UV-3000). Although the released LM was not confirmed to be intact by any other techniques such as electron microscopy, it was confirmed that the sampling solution has identical to diluted LM solution, without any interferences. The degradation of crosslinked HA gels by HAase was also examined in vitro. A slab (20 x 20 x 2 mm) of the crosslinked HA gel was immersed in 50 ml of 0. I4 M phosphate buffer solution at pH 7.4 (PBS) for 2 days in order to be equilibrated. Then, the degradation of crosslinked HA gel was initiated by immersing the gel into 50 ml of HAase-PBS solution at different concentrations at 37°C with stirring. For a long term experiment, the HAase-PBS solution was renewed every other day in order to prevent from the inactivation of HAase at environmental atmosphere. The degradation and LM release were estimated by the same manner as described above. The degradation of HA solution by HAase was examined by an intrinsic viscosity measurement [18]. In vivo inflammation responsive degradation of crosslinked HA gel A slab (approximately 20 x 10 x 2 mm ) of the crosslinked HA gel equilibrated by physiological saline solution was sterilized for 30 min in an autoclave ( 1.2 atm, 100°C ). No change in the HA content in the slab by the sterilization was confirmed previously, indicating that HA was not degraded during sterilization. The slab was surgically implanted subcutaneously in the dorsum of 5-week-old male Wistar rat (average body weight: 110 g). The incision (ca., 4 cm length) was sutured by surgical silk threads of 5 needle (Nescosuture@, Nippon Shoji Co., Tokyo, Japan) and then disinfected. At both 14 and 28 days after implantation, surgical injuries were made by incising (ca., 4 cm length ) of the dorsum skin to generate a preliminary inflammation reaction, which is known as a wound healing test [ 201. Furthermore, in order to quantify the

inflammation responsive degradation of crosslinked HA gels, granulomas were induced by harvesting p-carrageenin subcutaneously as an inflammation model: 2 wt.% carrageenin solution in physiological saline solution ( l-2 ml ) was injected subcutaneously in the vicinity of the implanted site after 2 weeks of implantation [ 2 11. The rat was sacrificed with excess amount of anesthetic 7 days after the wound healing test or subcutaneous injection of p-carrageenin was carried out. Then, the carrageenin-induced granuloma, and the implanted HA gel were excised. The residual amount of HA gel was determined by a carbazole-sulfuric acid method, which is a suitable procedure for the quantitative analysis of uranic acid [ 221. The uranic acid content in the subcutaneous tissue of dorsum in non-implanted rat was found to be negligible. Results and Discussion Limited and surface-controlled degradation of crosslinked HA gels by hydroxyl radicals The preparation of crosslinked HA gels by PGPGE has been previously reported [ 16 1. The preferred crosslinking reagents are multifunctional epoxides which can form ether linkages to HA molecules via hydroxyl groups. PGPGE is an unique crosslinker in terms of both water solubility and functionality of epoxy groups. In spite of its high water content, the crosslinked HA gel was mechanically tough and could be used as a slab. Figure 1 shows an SEM view of the surface of crosslinked HA gels. It was observed that LM was dispersed into the matrices of crosslinked HA gels. Average distance of LM dispersion was estimated to be around 0.5 pm. In our previous paper, it was clarified that crosslinked HA gels were specifically degraded by hydroxyl radicals [ 161. Figure 2 shows the representative results of limited degradation of a crosslinked HA gel in the presence of hydroxyl radicals. In this experiment, a slab of the crosslinked HA gel was immersed in a solution of Hz02 and then the addition of a certain amount of FeSO, solution with different concentration

137

Fig. 1. SEM view of lipid microspheres

I.,

4o

n

0

50

..a

100 Time (min)

in crosslinked HA gel.

*.

150

I 200

Fig. 2. Limited degradation of crosslinked HA gels by hydroxyl radicals. Hydroxyl radicals were generated by adding 1 ml of FeS04 solution with different concentrations into 100 ml of 5 mM Hz02 solution.

was repeated. Although stable in HzOz, the crosslinked HA gels showed rapid degradation within a short period after the addition of FeSO+ The rapid degradation is due to the rapid autoxidation of Fe*+ to Fe3+ by H202 in which hydroxyl radicals are produced by the following reaction: Fe2++H202+Fe3++OHo+OHThe reaction of HA with hydroxyl radicals has been reported to proceed preferentially via abstraction of the hydrogen on the carbon adjacent to the carboxyl group in the D-glucuronic acid unit, leading to glycosidic cleavage [ 23 1. Our results indicate that crosslinked HA gels can be degraded specifically by generated hydoxyl radicals

in the presence of other oxidizing agents and that the degradation can take place in pulse like (stimulus sensitive) way. In order to control the rate of drug delivery in response to the biological need of a patient, it should be possible to discontinue the degradation of the polymer matrix loaded with the drug when the biological need is eliminated. In this point of view, the degradation characteristics of the crosslinked HA gel in principle make it suitable for the application in an auto feed-back drug delivery system. The short life time of the hydroxyl radicals makes it difficult to examine the degradation kinetics of crosslinked HA gels. It has been reported that HA can form a complex with metal ions such as Cu*+ and Fe*+ and that this complex is very important to bring about a site-specific oxidation reaction in the presence of H202 [ lo- 13 1. Fe*+ appears to bind to HA which prevents the formation of hydroxyl radicals in the free solution, but hydroxyl radicals are still generated at a site-specific location on HA molecules [ 111. In this paper, the crosslinked HA gel was first immersed in FeSO, solution to form a HA-Fe*+ complex, and then moved to H202 solution for the generation of hydroxyl radicals (see experimental). This complexation has a possibility of altering the structure of the gel, however few change in the water content of the gel by the complexation was observed, because of using low FeSO, concentration ( 10 mM) . This complexation is considered to be very stable since the water content of the complexed gel was observed to be constant over a few days in PBS. Figure 3 shows a time course of the degradation of crosslinked HA gels at different H202 concentrations. The degradation time of these HA gels varied depending on the generation of hydroxyl radicals, every degradation occurred in proportion of time. Our previous paper demonstrated that degradation of crosslinked HA gels by hydroxyl radicals in this system took place preferentially at the surface [ 161. It is considered that H202 interfacing with a gel surface reacts with site-specific Fe*+ to generate hydroxyl radicals, leading to surface degradation of crosslinked HA gels. The kinetics of degradation of crosslinked HA gel was

138

theoretically estimated based on a surface degradation [ 1,16 1. If M, is the amount of polymer degraded from a slab of area A (both side of a slab) at any time t dMJdt = B.p*A

(1)

where B is the surface degradation rate, expressed in units of length/time, andp the density of the polymer matrix. The density of the gel expressed in units of weight/volume is included in equation ( 1) because dM,/dt is expressed in units of weight/time. The total mass of polymer can be calculated as follows. M, =phA/2

(2)

Fe2+conc ,.. 1OmM 0

20

40

60 80 Time (min)

100

1’20

Fig. 3. Degradation of crosslinked HA gels by hydroxyl radicals with different concentrations. The gels were immersed in 10 mM FeSO, solution for 2 days, followed by addition to solutions of HzOz with different concentrations ( 100 ml).

where Mm is the total amount of polymer and h the initial thickness of the slab. If t, is the time to complete degradation, the film thickness (h) can be expressed below. h = 2Bt, Equation

(3) ( 1) can be readily integrated

Ml = B-p-At

below. (4)

The combination of equation (4) with equations (2 ) and (3 ) gives an expression for surface-controlled degradation of polymers as follows [ 11. W/M,

= t/t,

(5)

All the data shown in Fig. 3 followed equation ( 5 ), the surface-controlled degradation of crosslinked HA gels by hydroxyl radicals. Figure 4 shows a time course of the degradation of crosslinked HA gels with the same weights but different shapes at a certain H202 condition. Here, two different shapes were used: one piece of slab and ten pieces of rectangular solids which were prepared from a slab with the same size as the former one. Although these HA gels reached complete degradation around the same time, their degradation profiles were quite different. Then, if assuming the rectangular solid as a cube, the equivalent expression for a cube to equation (5 ) is also introduced as follows [ 1, 16 1. M,/M,=l-[l-(t/t,)]3

(6)

2

4 Time (min)

6

Fig. 4. Degradation of crosslinked HA gels with different shapes by hydroxyl radicals. The gels were immersed in 10 mM FeSO, solution for 2 days, followed by addition to 5 mM HzOz solution (50 ml).

Equation

(5 ) yields another expression.

1 - [ 1 - (MJM,)

] l/3= t/t_

(7)

Figure 5 shows plots of equations (5 ) and (7) based on the data in Figs. 3 and 4. Plots of equation (7) from the data in Fig. 4 were situated below the theoretical line, as shown in Fig. 5. This may be due to the scale of rectangular solids used: each rectangular solid has a dimension of 0.34 x 0.26 x 0.50 cm, which is not a cube. Both plots provide almost a linear relationship, strongly suggesting that the degradation of crosslinked HA gels by hydroxyl radicals takes place via a surface-controlled mechanism. Figure 6 summarizes the degradation rate of

139

0.2

0

0.4

0.6

0.8

Fig. 5. Surface-controlled degradation profiles of crosslinked HA gels by hydroxyl radicals. All the plots were calculated based on Fig. 4.

8~10-~Fe*+conc.: 1OmM

o

1

2 3 4 5 H202 concentration (mM)

6

Fig. 6. Rates of the degradation of crosslinked HA gels by hydroxyl radicals. The degradation rate was calculated by equation ( 3 ) .

crosslinked HA gels, calculated from equation ( 3 ) . Degradation rate was expressed as a function of Hz02 concentration. Our previous paper demonstrated that the degradation rate of ethyleneglycol diglycidylether-crosslinked HA gels was around 10m4cm/s in the similar conditions of hydroxyl radical generation [ 16 1. Thus a variation of the degradation of crosslinked HA gels can be performed through both the design of crosslinking conditions and the generation of hydroxyl radicals. Degradation-controlled release of LM from crosslinked HA gels

Previously, the degradation of crosslinked HA gels by HAase was examined, and was clarified to be precluded by prevention from HAase ac-

cess into the gel [ 151. It was considered that intrusion of HAase into the HA gel must be strongly inhibited by this electrostatic repulsive force resulting in high tolerance of the HA gel toward HAase. Steric exclusion of HAase from crosslinked HA gels can be useful in the point of maintaining drug activity introduced in gel matrix. As any proteins cannot be intruded as much as HAase, drug activity in gel might be maintained until the degradation front reaches the drug. However, even if any protein intrusion into the HA gel is precluded, the degradation-controlled drug release from the HA gel cannot be guaranteed. In this point of view, it is suggested that any drug reservoirs should be required in order to release drug in proportion to the degradation of highly water swollen HA gel. As to the incorporation of drug into the crosslinked HA gel, a heterogeneous structure composed of drug microreservoir and degradable HA matrix can be proposed as a implantable device. In this sense, several biocompatible microparticles such as LM, albumin microspheres, and any other microcapsules, should be attractive [ 241, In order to not only confirm the feasibility of a heterogeneous HA gel for degradation-controlled drug release but also examine the stability of LM as a drug carrier in the heterogeneous device, LM was used in this paper. LM has been reported to be more stable than liposomes and used for parenteral nutrition in human. LM has been known to be taken up readily by phagocytic cells like liposomes [ 25 1, and has been recently utilized as a drug carrier for various lipophilic drugs [ 261. Figures 7 and 8 show release of LM in response to the degradation of crosslinked HA gels, corresponding to Figs. 3 and 4, respectively. These figures indicate that LM was released in proportion of the degradation of crosslinked HA gels (Figs. 3 and 4). In a similar manner to equation ( 5 ), if S, is the amount of microspheres released from the surface of a slab at any time t, surface-controlled release of LM can be introduced as follows.

wsx =tlt,

(8)

The equivalent expression of surface-controlled

140

80

0.8 -

7e Q) 60 3 1 2 40

8 0.6 v) rectangular solids

3 20 Fe2+conc.: 20

40

80 TimGeO(min)

1OmM 100

0

120

0.4

0.6

0.8

1.0

Mt/ Mm

Fig. 7. Lipid microsphere release from crosslinked HA gels degraded by hydroxyl radicals, This figure corresponds to Fig. 3. LM release was estimated by measuring the transmittance of the sampling solution at 500 nm using a visible-light spectroscopy.

80

0.2

Fig. 9. Lipid microsphere release from crosslinked HA gels in proportion to the degradation by hydroxyl radicals. All the plots were calculated based on Figs. 4 and 8.

.

‘;; o\ 13 60

.lo

DieCSS

Of

HAase conc.(u/ml):

10

n 213 A 13 l 0.13

20 Time (h)

0

2

4 Time (min)

6

Fig. 8. Lipid microsphere release from crosslinked HA gels with different shapes. This figure corresponds to Fig. 4. LM release was estimated by measuring the transmittance of the sampling solution at 500 nm using a spectroscopy.

release of microspheres scribed as follows. 1 - [ 1 - (S/S,)

] “j=

for a cube can be de-

Combination of equations equation ( 10) as follows. M,IKI

= S/SC

Fig. IO. Degradation of crosslinked HA gels by HAase with different concentrations. The degradation was estimated in HAase solutions with different concentrations in PBS at 37°C (50 ml).

linked HA gels was dependent on the surface area of degradable matrices of crosslinked HA gels. Degradation

t/t_=

30

of crosslinked HA gels by HAase

(9) (5) and (8) gives

(10)

At a similar manner, equations (7 ) and (9 ) also introduces equation ( 10). Plots of equation ( 10 ) based on the data of Fig. 8 are shown in Fig. 9. The close linear relationship in Fig. 9 indicates that LM were released in response to the matrix degradation. Thus, the release of LM from cross-

The degradation of crosslinked HAgels by HAase and their release of LM were also investigated. Figures 10 and 11 show the results of the degradation of the crosslinked HA gel by HAase and its responsive LM release, respectively. All the data in Figs. 10 and 11 provide a linear relationship between n/l,/M, and t/t, or S,/S, and t/t,. These results indicate that LM release was controlled by the surface degradation of the crosslinked HA gel. Figure 12 summarizes the degradation rate of crosslinked HA gels by HAase

141

In vivo degradation of crosslinked HA gels in response to inflammation

HAase conc.(u/ml):

20

10

n 213 A 13 l 0.13

30

Time(h) Fig. 11. Lipid microsphere release from crosslinked HA gels degraded by HAase. This figure corresponds to Fig. 10. 10-l G $10’2

solution 1

E S Q) 10-3. H

L/

.g 10 -4 Tii ‘0 c 1(-J-5F P

10-6’ 0.1

-.....’

1

. .-..‘.a

10

. .a.....(

100

..-I 1000

HAase concentration (u/ml) Fig. 12. Prevention of crosslinked HA gels by their enzymatic degradation. The degradation rate was calculated by equation (3).

from equation ( 3 ) . The degradation rate of crosslinked HA gels was far lower than that of non crosslinked HA, as shown in this ligure. This result indicates that the degradation was precluded by prevention from HAase access into the gel [ 161. Although the skin is known as the largest store of HAase, HAase generally presents in an inactive form. This is related to the regulation of metabolic exchanges by decreasing the viscosity of intracellular matrices [ 27,281. Thus the HAase concentration in the living body is believed to be much lower than used in the in vitro experiment. As the subcutaneous injected HA solution was reported to be degraded within a week [ 29 1, the enzymatic degradation of crosslinked HA gels after implantation will be negligible. calculated

A slab of the crosslinked HA gel was implanted subcutaneously in the dorsum of a rat, and the stability of the crosslinked HA gel at a subcutaneous site and the degradability of the HA gel by inflammation were examined. The crosslinked HA gels used in vivo were not incubated with FeSO, solution, since tissue inflammation produces hydroxyl radicals. Therefore, it must be stated that the system in vivo was not identical to those used in vitro. In our previous paper [ 161, it was found that approximately 20% of crosslinked HA gel was degraded within 10 days after the implantation but the residual gel was relatively stable over the period of 100 days. Such limited degradation during the first few days was considered to be due to the inflammation caused by a surgical incision for implantation. Then, in order to examine injury-dependent degradation of crosslinked HA gels, double surgical injuries were made every other week after the implantation: each injury was made by incising the dorsum skin for additional 4 cm length and then suturing. The result of degradation of crosslinked HA gels by additional surgical incisions are summarized in Fig. 13. It was found that the surgical incision at the dorsum induced degradation of the implanted HA gel to a certain extent, and the degradation was dependent on the number of surgical incisions. The difference in the degradation by surgical incisions was statistically significant (see Fig. 13 ) . Furthermore, p-carrageenin was injected subcutaneously in the vicinity of the implanted site Control (excised at 21-50 P.O.Day) Surgical incision x 1 (excised at 21-35 P.O.Day) Surgical incision x 2 (excised at 42 P.O.Day) I

0

.

.

.

.

1

20 40 60 Degradation of crosslinked HA gel (%)

Fig. 13. Limited degradation of crosslinked HA gels in response to preliminary inflammation in rats ( + S.E.M.).

142

References

Y

q

5.70X + 24.3 R2 = 0.878

Wet weight of granuloma (g) Fig. 14. Limited degradation of crosslinked HA gels in response to carrageenin-induced granulomas in rats.

in order to quantify the inflammation responsive degradation of crosslinked HA gels. Granulomas are well known as focal, predominantly mononuclear tissue inflammations evoked by persistent irritants. Phagocytic cells such as macrophages and their products are assumed to be involved in the development of granulomatous formations [ 301. The p-carrageenin granulomas are reported to grow rapidly reaching maximum wet weight in 5 days [ 3 11. Thus, the wet weight of granulomas after 5 days of the carrageenin injection was measured as the degree of local tissue inflammations. Figure 14 shows the relation of carrageenin-induced granulomas and degradation of crosslinked HA gels in rat. The degradation of crosslinked HA gels was well correlated with the wet weight of carrageenin-induced granulomas. These results indicate that the crosslinked HA gel was degradable in response to inflammation although the gel was not afftected under normal healthy conditions.

Acknowledgements The authors are grateful to Prof. Tamotsu Kondo, Miss Toshiko Yamazaki, Department of Pharmaceutical Sciences, Science University of Tokyo, for their collaboration in a part of this study. Thanks are due to Dr. Glen Kwon, International Center for Biomaterials Sciences, Tokyo Women’s Medical College, for his valuable discussions.

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