DOI: 10.1148/radiol.2361040669
(Radiology 2005;236:151-158.)
© RSNA, 2005
Chitin-based Embolic Materials in the Renal Artery of Rabbits: Pathologic Evaluation of an Absorbable Particulate Agent1
Byung Kook Kwak, MD,
Hyung Jin Shim, MD,
Sang-Mun Han, PhD and
Eon Sub Park, MD
1 From the Departments of Radiology (B.K.K., H.J.S.) and Pathology (E.S.P.), Yongsan Hospital, Chung-Ang University College of Medicine, 65-207 Hangangro-3-Ga, Yongsan-Gu, Seoul 140-757, Korea; and Department of Environmental Health, Seoul Health College, Gyeonggi-Do, Korea (S.M.H.). Received April 12, 2004; revision requested June 22; revision received August 1; accepted August 18. Supported by Korea Research Foundation Grant KRF-99-042-F00125.
Address correspondence to B.K.K. (e-mail: kwakbk{at}cau.ac.kr).
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ABSTRACT
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PURPOSE: To prospectively evaluate the tissue reaction to and the embolic effect and absorption of chitin and chitosan microspheres and polyvinyl alcohol (PVA) in the renal artery of rabbits.
MATERIALS AND METHODS: This experiment was performed in accordance with regulations on animal care and experiments. Thirty-six New Zealand white rabbits were divided into four groups according to the materials (PVA, chitin particles, and chitosan particles, and chitosan microspheres; diameter, 150–250 µm) used for embolization of the right renal artery. A rabbit from each group was sacrificed 1 and 3 days and 1, 2, 4, 8, 16, 24, and 32 weeks after embolization. Gross and microscopic pathologic findings were examined with hematoxylin-eosin, Masson trichrome, and Victoria blue staining.
RESULTS: Gross pathologic findings were examined, and swelling of embolized kidneys was observed 1 and 3 days after embolization, whereas shrinkage of the embolized kidneys was consistently seen after 2 weeks, with a hard consistency and nodular surfaces being noted. At histologic analysis, chitosan microspheres filled the lumen more compactly than did other particles. With PVA, a large amount of capillary formations occurred within the embolized arteries, whereas chitin particles and chitosan microspheres showed a lower rate of capillary formation. The shape of all embolic materials remained intact until week 8, at which time the materials gradually decreased in size and number. The chitosan particles and the chitosan microspheres were absorbed around weeks 16 and 24, respectively.
CONCLUSION: Chitosan microspheres have great potential as a new embolic material since they block blood vessels more compactly with a lower rate of capillary formation. This material is biocompatible, and it is absorbed 24 weeks after embolization.
© RSNA, 2005
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INTRODUCTION
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For safe and effective embolotherapy, the selection of an appropriate embolic material is essential. The requirement of the material can vary according to the condition of the lesion or the disease that is being treated. Consideration must be given to whether the embolization is to be made permanent or temporary and whether the blood vessel to be embolized is an end artery or an artery with collateral blood flow. Because there is no ideal embolic material that is applicable to all situations, the material should be selected specifically for each situation. Thus, various characteristics of embolic materials, including the time necessary for the material to be absorbed, the degree of penetration into the blood vessels, and the size and shape of the particles should also be considered.
The particulate embolic materials that are widely used are gelatin sponge and polyvinyl alcohol (PVA). Gelatin sponge is absorbed between 7 and 21 days (1); subsequently, it is used for temporary embolization. On the other hand, because PVA is known to be nonbiodegradable, it is regarded as a permanent embolic material (2). A fraction of particle embolic material with the same size is obtained by using a sieve filtering process. Because PVA particles have irregular shapes, even large particles may pass through a fine sieve if they are oriented properly; thus, the sizes of the particles can be inconsistent (3,4). In addition, the PVA particles tend to aggregate, which causes occlusion of the catheters and proximal artery instead of occlusion of the desired blood vessels (3–5). The tris-acryl gelatin microsphere (Embosphere Microsphere; Biosphere Medical, Rockland, Mass) appears to have the characteristics of a globe-shaped nonabsorbent embolic material. The material is evenly sized, is deformable, and does not aggregate (6,7). Because of these characteristics, tris-acryl gelatin microspheres more actively cause distal vascular penetration, and they are more effective and cause less bleeding than PVA for preoperative blockade (8).
Chitin, a poly(ß-[1,4]-N-acetyl-D-glucosamine), is the second most abundant natural biopolymer after cellulose, and it is abstracted from the crust of crabs and shrimp. Chitosan is a poly(ß-[1,4]-D-glucosamine) that can be prepared by N-deacetylation of chitin, and it is known as material that is highly biocompatible and biodegradable. Furthermore, chitosan has low toxic and antigenic properties (9,10). Because of these characteristics, chitosan has drawn attention in the medical field as a useful material. It is easily formed into a globe-shaped fine particle, and it has the qualities of an embolic material. To our knowledge, however, no research has been conducted on the absorption and tissue reaction after embolization when chitosan is used as an embolic material. Thus, the purpose of our study was to prospectively evaluate the tissue reaction to and the embolic effect and absorption of the chitin and chitosan microspheres and the PVA in the renal arteries of rabbits.
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MATERIALS AND METHODS
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The present experiment was performed according to Chung-Ang University Hospital regulations on animal care and experiments. Thirty-six New Zealand white rabbits were divided into four groups, with nine rabbits in each group. In group 1, PVA with a 150–250-µm diameter (Contour; Boston Scientific, Freemont, Calif) was used as the embolic material in the renal artery. In groups 2, 3, and 4, 150–250-µm chitin particles, 99% deacetylated 150–250-µm chitosan particles, and 75% deacetylated 150–250-µm chitosan microspheres were used as the embolic materials, respectively.
Preparation of Embolic Materials
To produce chitin, calcium carbonate was removed from the crust of king crabs (C & C Science, Gyeonggi-Do, Korea) by depositing their shells in a 5% hydrochloric acid solution for 24 hours. After this time, the remaining hydrochloric acid was washed away with water. Protein was then removed from the shells by boiling them in a 5% sodium hydroxide solution for 5 hours. In addition, the pigment was removed by using 0.5% potassium permanganate, and the manganese content was reduced by using 0.1% oxalic acid at 60°–70°C. The remaining parts of the shells were washed to produce pure white chitin. The chitin particles were made by grinding the shells into powder against a mesh filter with 250-µm holes. The 99% deacetylated chitosan particles were then prepared by dipping the chitin particles into a 50% sodium hydroxide solution at 121°C for 1 hour, and the same procedure was repeated five times. Next, 4 g of 75% deacetylated chitosan was dissolved in 100 mL of acetic acid, from which impurities were removed with a filtering process. Finally, fine drops of 75% deacetylated chitosan microspheres were produced with a custom-made microsphere producer by dropping the chitosan particles into a coagulating solution (sodium hydroxide-ethanol–distilled water, 4/20/76, W/V/V). The embolic materials were prepared by one of the authors (S.M.H.).
Microscopic Comparison
The 150–250-µm PVA particles were used for comparison, while the chitin and chitosan embolic materials were obtained by filtering the particles and microspheres through an overlaid standard 150–250-µm sieve (Chunggye, Seoul, Korea). A total of 100 particles of each sample were examined with a stereomicroscope (BX60 F5; Olympus Optical, Tokyo, Japan), and their lengths were measured by using an image analyzer (Image-Pro Plus 4.51; Media Cybernetics, Silver Spring, Md).
Angiography and Embolization
Embolization of a renal artery was performed in 36 New Zealand white rabbits that weighed 2.0–3.5 kg each. The results of laboratory blood analysis, including complete blood cell count with differential count, blood chemistry (blood urea nitrogen, creatinine, aspartate aminotransferase, and alanine aminotransferase), and electrolytes (sodium, potassium, and chlorine), were normal before this study was initiated. A 35-mg per kilogram of body weight dose of ketamine (Ketalar; Yuhan Yanghang, Seoul, Korea) and a 5 mg/kg dose of xylazine (Rompun; Bayer Korea, Seoul, Korea) were injected intramuscularly for anesthesia.
One rabbit in the chitosan microsphere group (group 4) died of an anesthesia-related cause before embolization. A 4-F angiography cobra catheter (Terumo, Tokyo, Japan) was inserted through the right femoral artery, and the right renal artery was selected as the test target. Renal angiography was performed with injection of 7 mL of contrast material (Omnipaque 300; Amersham, Oslo, Norway) to enable identification of the right renal artery and renal parenchyma. Each embolic material was injected with a 5-mL syringe into the selected right renal artery by means of fluoroscopy until the artery was totally occluded. When excessive embolic material was injected, it flowed back into the left renal artery and killed the rabbit. In the chitin group, one rabbit died 4 days after embolization because the reflux of embolic material into the left renal artery caused an occlusion of both renal arteries. Subsequent injections were performed with even more careful attention given to injection of the embolic material. After embolization, the catheter was removed, and the femoral artery was tied with 3–0 silk. A 100 mg/kg dose of ceftezole sodium (Shin Poong, Seoul, Korea) was injected intramuscularly once a day for 3 days after embolization to prevent infection. Procedures were performed concurrently by two interventionalists (B.K.K., H.J.S.).
Pathologic Evaluation
A rabbit from each group was randomly sacrificed 1 and 3 days and 1, 2, 4, 8, 16, 24, and 32 weeks after embolization. All the kidneys, including the embolized right kidneys and the normal left kidneys, were harvested and cut along the sagittal plane into halves. Gross pathologic examination focused on the consistency, surface, discoloration, and size of the kidneys and was performed by one of the authors (B.K.K.). The harvested kidneys were first fixed with 10% formalin for 24 hours and then stained with hematoxylin-eosin. Kidneys of rabbits in groups 1, 2, and 4 were additionally stained with Masson trichrome and Victoria blue to examine the degree of organization and the fibrosis occurring inside the renal artery, as well as the degree of destruction of the elastic lamina on the vessel wall. The pathologic findings of all the specimens were evaluated by a blinded pathologist by means of light microscopy, and the results were independently confirmed by another blinded pathologist (E.S.P.).
All pathologic findings were graded as negative, slightly reactive, moderately reactive, or substantially reactive according to the degree of reaction, which is based on the following morphologic findings: inflammation, organization, formation of capillaries, reaction of foreign bodies, resorption of embolic material, fibrosis, and destruction of internal elastic lamina of the blood vessels in the renal hilum. Organization is defined as the proliferation of cells with a subintimal origin in the vascular lumen within the embolized kidneys. The formation of capillaries is defined as the newly developed vascular structure within the embolized vessel lumen.
The resorption of embolic material was graded as slightly or moderately reactive according to the absorption deformity or size reduction of the embolic material and the frequency of detection of the embolic material within the embolized renal hilar artery. It was graded as substantially reactive when the embolic material was absorbed completely. The degree of fibrosis, which was determined with Masson trichrome staining, was graded as slightly reactive when fibrosis was found slightly around blood vessels, substantially reactive when fibrosis grew thick and was found in the organizing materials of the embolized vessels, or moderately reactive when fibrosis formation was moderate. The degree of destruction of elastic fibers of the blood vessels, which was determined with Victoria blue staining, was digitized with the Schwartz method (11). The degree was determined to be negative when the internal elastic lamina was normal, slightly reactive when the internal elastic lamina was destroyed with the intact elastic fiber in the middle membrane, moderately reactive when the internal elastic lamina and the elastic fiber in the middle membrane were destroyed with the intact external elastic lamina, or substantially reactive when elastic fibers in the three layers of the blood vessels were all destroyed (11).
Blood Analysis and Chemistry
Before rabbits were sacrificed, blood was sampled, and the complete blood cell count with the differential count and basic blood chemistry (blood urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, electrolytes, etc) were evaluated. The standards of Yu et al (12) and Wolford et al (13) were used as criteria to determine normal ranges.
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RESULTS
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Embolic Materials
According to light microscopic findings, the PVA particles had irregular shapes, uneven surfaces, and unequal sizes. On the other hand, chitin and chitosan particles were mostly thin scale-shaped plates. Chitosan microspheres were globular or elliptical in shape and had a uniform size (Fig 1). The length of PVA particles was 326 µm ± 89.1 (mean ± standard deviation), while the length of chitin particles, chitosan particles, and chitosan microspheres was 335 µm ± 56.8, 466 µm ± 100.2, and 271 µm ± 37.2, respectively (Fig 2).
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Figure 1a. Light microscopic findings of each embolic material. (a) PVA particles with irregular shape, (b) chitin particles and (c) chitosan particles with thin scale-shaped plates,and (d) chitosan microspheres with globular or elliptic form.
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Figure 1b. Light microscopic findings of each embolic material. (a) PVA particles with irregular shape, (b) chitin particles and (c) chitosan particles with thin scale-shaped plates,and (d) chitosan microspheres with globular or elliptic form.
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Figure 1c. Light microscopic findings of each embolic material. (a) PVA particles with irregular shape, (b) chitin particles and (c) chitosan particles with thin scale-shaped plates,and (d) chitosan microspheres with globular or elliptic form.
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Figure 1d. Light microscopic findings of each embolic material. (a) PVA particles with irregular shape, (b) chitin particles and (c) chitosan particles with thin scale-shaped plates,and (d) chitosan microspheres with globular or elliptic form.
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Figure 2a. Size distribution of (a) PVA particles and (b) chitosan microspheres. A total of 100 PVA particles (150–250 µm) show a wider distribution in size than 100 chitosan microspheres (150–250 µm).
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Figure 2b. Size distribution of (a) PVA particles and (b) chitosan microspheres. A total of 100 PVA particles (150–250 µm) show a wider distribution in size than 100 chitosan microspheres (150–250 µm).
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Embolization
One rabbit died of an anesthesia-related cause, and one died of reflux of embolic material in the left renal artery. Embolization was successfully performed in the remaining 34 rabbits, and none of them died because of a postembolization complication. There were nine rabbits in both group 1 and group 3 and eight rabbits in both group 2 and group 4. In all four groups, the embolic material was easily injected through the catheter without causing any blockage.
Gross Pathologic Findings
Severe proliferations of the blood vessels by the retroperitoneal fat around the embolized kidney were observed from day 1 to week 1 in groups 1, 2, and 4 and from day 3 to week 2 in group 3. Swelling of the embolized kidneys, which was probably caused by edema from cytotoxicity, was observed 1 and 3 days after embolization in groups 1, 2, and 3, and up to 1 week after embolization in group 4. The size of the embolized kidneys decreased afterward. The normal left kidneys showed substantial compensatory hypertrophy after week 4 in all groups. Because of the ischemic changes, the dark brown color of the embolized kidneys on day 1 gradually paled. After 2 weeks, the color was pink, and we observed the hard consistency and nodular surfaces of the kidney. After the blood vessels of the retroperitoneal fat disappeared, irregularly shaped small blood vessels were scattered in only the shrunken kidney. The shrinkage of the embolized kidneys was observed from week 2 in group 1 and from week 4 in groups 2, 3, and 4. As a whole, there was no substantial difference in gross observations among the four groups.
Microscopic Findings
The renal arteries evaluated in this study were the segmental arteries at the hilum. PVA particles were seen as irregular and pointed structures, with a pale pink-blue color at hematoxylin-eosin staining, a dark brown color at Masson trichrome staining, and a dark blue color at Victoria blue staining. It should be noted, however, that PVA particles stained poorly with Victoria blue. Meanwhile, both chitin particles and chitosan particles and microspheres appeared red at hematoxylin-eosin staining, although chitosan particles and microspheres appeared to be darker. Chitosan particles and microspheres appeared dark red at Masson trichrome staining, and they appeared to be pale pink at Victoria blue staining. Chitin particles appeared dark blue at Masson trichrome staining and pale pink at Victoria blue staining.
Chitin and chitosan particles were long and rod shaped, whereas the chitosan microspheres were uniformly globe shaped. The filling pattern of the blood vessels was different depending on the shape of embolic materials. Each embolic material filled the vessel lumen, and there were spaces among the embolic materials. The spaces were most prominent among the thin rod shaped chitin particles, which were followed in prominence by the irregularly shaped PVA particles. Although chitosan particles were also thin and rod-shaped, they bent and folded within the vessel; as a result, the spaces among the chitosan particles were not as large as those among chitin particles. On the other hand, the chitosan microspheres showed a compact filling of the vessel lumen, and this was a consequence of having much smaller empty spaces among them (Fig 3).
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Figure 3a. Photomicrographs of rabbit hilar renal arteries filled with various embolic materials. (a) PVA particles and (b) chitin particles viewed 3 days after embolization. (c) Chitosan particles viewed 1 day after embolization. (d) Chitosan microspheres viewed 3 days after embolization. The chitosan microspheres filled the lumen more compactly than the other particles. (Hematoxylin-eosin stain; original magnification, x40.)
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Figure 3b. Photomicrographs of rabbit hilar renal arteries filled with various embolic materials. (a) PVA particles and (b) chitin particles viewed 3 days after embolization. (c) Chitosan particles viewed 1 day after embolization. (d) Chitosan microspheres viewed 3 days after embolization. The chitosan microspheres filled the lumen more compactly than the other particles. (Hematoxylin-eosin stain; original magnification, x40.)
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Figure 3c. Photomicrographs of rabbit hilar renal arteries filled with various embolic materials. (a) PVA particles and (b) chitin particles viewed 3 days after embolization. (c) Chitosan particles viewed 1 day after embolization. (d) Chitosan microspheres viewed 3 days after embolization. The chitosan microspheres filled the lumen more compactly than the other particles. (Hematoxylin-eosin stain; original magnification, x40.)
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Figure 3d. Photomicrographs of rabbit hilar renal arteries filled with various embolic materials. (a) PVA particles and (b) chitin particles viewed 3 days after embolization. (c) Chitosan particles viewed 1 day after embolization. (d) Chitosan microspheres viewed 3 days after embolization. The chitosan microspheres filled the lumen more compactly than the other particles. (Hematoxylin-eosin stain; original magnification, x40.)
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Inflammation was seen most frequently in the renal arteries embolized with chitin particles. Inflammation was seen less frequently in renal arteries embolized with chitosan particles and chitosan microspheres, and the least inflammation was seen in renal arteries embolized with PVA particles. Meanwhile, the organization and formation of the capillaries and fibrosis often coincided; they were present together to the same degree, and these phenomena were observed most often in the arteries embolized with PVA particles and followed in frequency by arteries embolized with chitosan particles, chitosan microspheres, and chitin particles (Fig 4).
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Figure 4a. Photomicrographs show the formation of capillaries and fibrosis in the embolized arteries. Numerous capillaries (a, substantially reactive, arrows) (hematoxylin-eosin stain; original magnification, x200) filled with red blood cells and blue fibrosis (c, moderately reactive, arrowheads) (Masson trichrome stain; original magnification, x100) are seen in the organized artery 4 weeks after PVA embolization. In contrast, chitosan microspheres show less capillary formation (b, slightly reactive) (hematoxylin-eosin stain; original magnification, x200) and fibrosis (d, slightly reactive) (Masson trichrome stain; original magnification, x100) 4 weeks after embolization.
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Figure 4b. Photomicrographs show the formation of capillaries and fibrosis in the embolized arteries. Numerous capillaries (a, substantially reactive, arrows) (hematoxylin-eosin stain; original magnification, x200) filled with red blood cells and blue fibrosis (c, moderately reactive, arrowheads) (Masson trichrome stain; original magnification, x100) are seen in the organized artery 4 weeks after PVA embolization. In contrast, chitosan microspheres show less capillary formation (b, slightly reactive) (hematoxylin-eosin stain; original magnification, x200) and fibrosis (d, slightly reactive) (Masson trichrome stain; original magnification, x100) 4 weeks after embolization.
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Figure 4c. Photomicrographs show the formation of capillaries and fibrosis in the embolized arteries. Numerous capillaries (a, substantially reactive, arrows) (hematoxylin-eosin stain; original magnification, x200) filled with red blood cells and blue fibrosis (c, moderately reactive, arrowheads) (Masson trichrome stain; original magnification, x100) are seen in the organized artery 4 weeks after PVA embolization. In contrast, chitosan microspheres show less capillary formation (b, slightly reactive) (hematoxylin-eosin stain; original magnification, x200) and fibrosis (d, slightly reactive) (Masson trichrome stain; original magnification, x100) 4 weeks after embolization.
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Figure 4d. Photomicrographs show the formation of capillaries and fibrosis in the embolized arteries. Numerous capillaries (a, substantially reactive, arrows) (hematoxylin-eosin stain; original magnification, x200) filled with red blood cells and blue fibrosis (c, moderately reactive, arrowheads) (Masson trichrome stain; original magnification, x100) are seen in the organized artery 4 weeks after PVA embolization. In contrast, chitosan microspheres show less capillary formation (b, slightly reactive) (hematoxylin-eosin stain; original magnification, x200) and fibrosis (d, slightly reactive) (Masson trichrome stain; original magnification, x100) 4 weeks after embolization.
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Giant cell reaction, which was due to embolized foreign materials, appeared prominently 1–2 weeks after embolization, and it lasted until week 32. The degree of reaction was lowest with chitosan microspheres. All embolic materials maintained their shape until week 8. PVA particles used in embolization showed a progressive decrease in size and number within the vessel lumina from week 8 to week 32 (Table 1). At week 24, the fragmentation of PVA particles accompanied by the formation of abundant foreign body giant cells suggested the absorption of PVA particles (Fig 5). Chitin particles showed fragmentation and absorption at week 24, and this was accompanied by foreign body giant cell reaction; however, the chitin was not absorbed completely until week 32. This finding was similar to findings seen with PVA particles (Table 2). Chitosan particles first showed fragmentation and absorption at week 16, and they were absorbed completely by week 24 (Table 3). Absorption of chitosan microspheres was initially noted at week 24, and it was accompanied by foreign body giant cell reaction at microscopic examination. The globe shape became irregular, and it had a moth-eaten appearance (Fig 5). At week 32, chitosan microspheres were absorbed completely, and they were not found within vessel lumina (Table 4).
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Figure 5a. Photomicrographs show the absorption of embolic materials. At week 24, the fragmentation of PVA particles, accompanied by the formation of abundant foreign body giant cells, suggests the absorption of PVA particles (a, slightly reactive). (Hematoxylin-eosin stain; original magnification, x100.) The absorption of chitosan microspheres is seen at week 24 and is accompanied by foreign body giant cell reaction; the globe shape becomes irregular and takes on a moth-eaten appearance (b, slightly or moderately reactive). (Hematoxylin-eosin stain; original magnification, x100.)
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Figure 5b. Photomicrographs show the absorption of embolic materials. At week 24, the fragmentation of PVA particles, accompanied by the formation of abundant foreign body giant cells, suggests the absorption of PVA particles (a, slightly reactive). (Hematoxylin-eosin stain; original magnification, x100.) The absorption of chitosan microspheres is seen at week 24 and is accompanied by foreign body giant cell reaction; the globe shape becomes irregular and takes on a moth-eaten appearance (b, slightly or moderately reactive). (Hematoxylin-eosin stain; original magnification, x100.)
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At Victoria blue staining, vascular injuries appeared to become more severe with the passing of time in three of the groups. The degree of vascular injuries was moderately reactive when PVA particles and chitosan microspheres were used and substantially reactive when chitin particles were used (Tables 1, 2, 4; Fig 6). There was, however, no hemorrhage or extravasation for any of the embolic materials.
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Figure 6a. Photomicrographs show vascular injury. The internal elastic lamina of the hilar renal arteries in rabbits embolized with (a) PVA particle and (b) chitosan microspheres 4 weeks after embolization show moderately reactive and negative pathologic findings in destruction, respectively. (Victoria blue stain; original magnification, x200.)
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Figure 6b. Photomicrographs show vascular injury. The internal elastic lamina of the hilar renal arteries in rabbits embolized with (a) PVA particle and (b) chitosan microspheres 4 weeks after embolization show moderately reactive and negative pathologic findings in destruction, respectively. (Victoria blue stain; original magnification, x200.)
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Blood Analysis and Chemistry
For the blood cell analysis, no substantial increase or decrease was found among the four groups. In particular, an increase of the eosinophil count, which may have been caused by an allergic reaction, was not seen. For the blood chemical analysis, there was no substantial increase in the group embolized with chitin and the group embolized with chitosan compared with the group embolized with PVA particles.
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DISCUSSION
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Chitin is a natural biopolymer obtained from the exoskeleton of crustaceans, and chitosan is obtained mainly from the deacetylation of chitin. Because chitosan is very weakly antigenic and highly biocompatible, it has drawn attention for use as a material of medical devices (9,10). The safety of various kinds of chitin and chitosan as embolic materials was demonstrated in this experiment, as none of the rabbits died of causes related to the embolic material. Furthermore, no substantial abnormality was observed at hematologic or blood chemical examination when these various chitin and chitosan embolic materials were compared with PVA. A toxicologic examination that was performed by an outside institution also supported the compatibility and safety of chitin and chitosan.
Reports (3,4,7,8) have shown the merits of microspherical embolic material in comparison with irregular-size materials. PVA particles, which are irregular in shape and size, frequently clog either the catheter or the proximal portions of the vessels that physicians wish to block. This can result in reopening of the embolized vessels. In contrast, because the tris-acryl gelatin microsphere has a spherical shape, it is easy to deliver material of a specific size to a desired area of distribution. The uniform size and spherical shape of the material make it possible for the material to penetrate deeper into blood vessels, and the material will not pass through the capillary (7,8). This study also demonstrated that the PVA particles were more widely distributed than the chitosan microspheres of the same size (Fig 2). In addition, an advantage of such a globe-shaped embolic material is that it can fill the vessel lumen more compactly than PVA or the long rod-shaped particles (Fig 3).
In the embolization of the blood vessel, a certain degree of inflammation can increase the effect of embolization. Subsequently, an embolic material that causes inflammation may not be a disadvantage; however, because excessive inflammatory reaction may cause the destruction of a vessel wall, the embolic material may leak outside the blood vessel (14). Such an inflammation is known to occur from an allergic reaction to the direct toxic effect of ischemia and the embolic material itself (15–17). Although the chitin and chitosan used in this experiment caused more inflammation than did PVA particles, it was not severe enough to destroy the vessel wall (Tables 1–4, Fig 6).
Generally, the organization and formation of the capillaries and fibrosis appeared to occur and coincide with one another to the same degree (18,19). Previous reports (2) have shown the formation of capillaries composed of only endothelial cells without a smooth muscle layer or other vessel elements inside the blood vessel. Our study also demonstrated that these phenomena occurred at the same time in each group, regardless of the use of different embolic materials (Tables 1, 2, 4; Fig 4).
In this experiment, the formation of capillaries was observed most frequently with PVA particles, followed by chitosan particles and chitosan microspheres. Formation of capillaries was observed least frequently with chitin particles. Greater formation of capillaries in the presence of PVA particles after embolization can be interpreted as a reopening of the embolized artery or the recurrence of a lesion. On the other hand, use of chitosan microspheres usually is associated with poor capillary formation, compact filling of the lumen, and deeper penetration of the microspheres in comparison with PVA particles. It is remarkable that chitin showed the least amount of capillary formation among the embolic materials, in spite of the large empty spaces. This suggests a possible separate relationship for the formation of capillaries for each material.
Because PVA is not biodegradable in the human body, it has been regarded as a permanent embolic material. According to research by Davidson and Terbrugge (2), the fragmentation of PVA particles was not observed in tissue obtained from a facial vascular malformation 8 years after embolization; in fact, there was only a little calcification present. Other researches, however, indicate that embolic materials were not regularly observed after embolization (20), and different reports insisted that embolic materials were not observed, possibly because of distal migration (15,16). In this study, however, PVA particles were heavily accompanied by foreign body giant cells in the blood vessel of a rabbit's renal hilum from week 1 to week 32, and the number and size of PVA particles decreased from week 8 to week 32. Foreign body giant cells do not indicate toxicity by themselves, but they do represent a marker of poor biocompatibility of some artificial materials (21,22). At week 24, because fragmentation or deformity of PVA particles was accompanied by the formation of abundant foreign body giant cells, the possibility of the absorption of PVA particles is strongly suggested (Fig 5). Thus, after interpretation of our findings, we cautiously suggest the possibility that PVA particles are partially absorbable after week 8.
In the majority of animals in which chitin and chitosan embolic materials were used in this experiment, foreign body giant cells appeared 1–2 weeks after embolization, and these cells were observed up to week 32. We began to observe foreign body giant cells even before absorption of the material was confirmed with microscopic examination, and the giant cells were continuously observed, even after the embolic material was absorbed. To our knowledge, the use of chitosan as an embolic material has not been studied; therefore, there is no report on its mechanism of absorption inside the blood vessels. In addition, there are only a few reports on the extravascular biodegradation of chitin and chitosan in vivo (23). Although the mechanism of the in vivo absorption of chitin and chitosan has not been clearly explained, polysacchrides are generally degraded by enzymatic hydrolysis. Since both chitin and chitosan are kinds of carbohydrate, they are expected to follow the fate of most polysacchrides. The most effective hydrolysis enzyme is lysozyme. The enzyme exists in all parts of the body (23). It is assumed that lysozyme secreted in relation to a foreign body giant cell reaction cuts a polymer into fine pieces, and these pieces are in turn absorbed through phagocytosis (24). Chitin particles and chitosan microspheres were observed along with the foreign body giant cell reaction at week 24, while chitosan particles were absorbed by week 16. The chitin and chitosan embolic materials were fragmented and deformed, and this observation supports the idea that the mechanism of absorption for the materials operates through a process of lysis and phagocytosis by the foreign body giant cells.
In addition, it has been known that the in vivo absorption of chitin and chitosan embolic materials differs according to the degree of the material's deacetylation. Tomihata and Ikada (23) reported chitin and 68.8% deacetylated chitosan were absorbed faster than 73.3% deacetylated chitosan when they were implanted in a rat's subcutaneous tissue. In contrast to their results, our study showed that chitin was the least absorbed material, and on the basis of the degree of deacetylation, the 75% deacetylated chitosan microsphere was absorbed less than the 99% deacetylated chitosan particle. The causes of such differences are still unknown, but they are assumed to be due to the differences of absorption by the subcutaneous tissue and blood vessels, as well as the differences in molecular weight of the materials. Further research on this matter is necessary, but it is thought that various embolic materials can be created with different absorption time. It may also be possible to control the absorption time by controlling the degree of the deacetylation of chitin and chitosan.
Meanwhile, at Victoria blue staining, vascular injuries appeared to become more severe over time for all groups. The proposed mechanisms for the destruction of the elastic lamina include ischemia, direct toxic effects of embolized materials, or focal angionecrosis by embolized materials to the vessel wall (2,11,21). Although hemorrhage might be expected to occur as a result of vessel necrosis, bleeding has rarely been reported as a complication of embolization with each embolic material (16). In this study, there was no hemorrhage or extravasation of embolic materials.
There were some limitations of this study. The first limitation was that the results did not lend themselves to statistical analysis because a rabbit from each group was sacrificed at a given time. The second limitation was that this study did not include analysis of the peripheral blood vessels or the renal parenchyma. In fact, only the segmental artery of the renal hilum was included. However, we were also compelled to investigate tissue reaction and the absorption of embolic materials in the peripheral blood vessels and renal parenchyma to accurately analyze the whole embolic effect. The third limitation was that other spherical agents were not included for comparison. However, they were not available at the time our study was performed.
Practical applications: We report that chitosan microspheres have potential as a new embolic material, since they block the blood vessels more compactly and with a lower rate of capillary formation than PVA particles. Furthermore, these new materials have excellent biocompatibility, and the fact that they are absorbed about 24 weeks after an embolization procedure makes them excellent candidates for future studies and appropriate clinical use as a new absorbable particulate agent.
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FOOTNOTES
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Abbreviations: PVA = polyvinyl alcohol
Authors stated no financial relationship to disclose.
Author contributions: Guarantors of integrity of entire study, B.K.K., S.M.H.; study concepts and design, B.K.K., H.J.S.; literature research, B.K.K., S.M.H.; experimental studies, S.M.H., B.K.K.; data acquisition, S.M.H., B.K.K.; data analysis/interpretation, E.S.P., B.K.K.; manuscript preparation, B.K.K., H.J.S.; manuscript definition of intellectual content, all authors; manuscript editing, B.K.K., S.M.H.; manuscript revision/review, all authors; manuscript final version approval, B.K.K.
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References
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ABSTRACT
INTRODUCTION
