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Reduction of adhesion formation with hyaluronic acid after peritoneal surgery in rabbits
F E R T I L I T Y AND S T E R I L I T Y ®
Vol. 67, No. 3, March 1997
Cop:~ight ~ 1997 American Society for Reproductive Medicine
Printed on acid-free paper in U. S. A.
Reduction of adhesion formation with hyaluronic acid after peritoneal surgery in rabbits*
Kathleen E. Rodgers, Ph.D.t$ Douglas B. Johns, Ph.D.§ Wefki Girgis, D.V.M.t
Joseph Campeau, M.S.t Gere S. diZerega, M.D.t
University of Southern California School of Medicine, Los Angeles, California, and Ethicon, Inc., Somerville, New Jersey
Objective: To examine the effect ofhyaluronic acid, a high-molecular-weight glucosaminoglycan found in the extracellular matrix, on the formation of adhesions, a major source of postoperative complications. Design: The ability of hyaluronic acid to reduce adhesion formation was evaluated using a standardized rabbit model. The material was administered IP at the end of surgery. Setting: University laboratory. Animal(s): New Zealand White female rabbits. I n t e r v e n t i o n ( s ) : Intraperitoneal administration of various formulations of hyaluronic acid at the end of surgery. Main O u t c o m e Measure(s): One week after surgery, a second laparotomy was performed and the extent of adhesion formation was determined. Result(s): Five separate molecular weight ranges ofhyaluronic acid representing eight viscosities between 1,000 and 12,000 centipoise (CPS) were shown to reduce adhesion formation in this model. All volumes, 1 to 30 mL, of hyaluronic acid tested reduced adhesion formation. In addition, the low-viscosity, low-molecular-weight hyaluronic acid significantly reduced adhesion formation when added to the trauma site or when injected at a site remote from the trauma area. Conclusion(s): This study showed that hyaluronic acid administered at the end of surgery reduced adhesion formation. Fertil Steril ® 1997;67:553-8 Key Words: Hyaluronic acid, adhesion, intraperitoneal, viscoelastic
A d h e s i o n f o r m a t i o n a f t e r p e r i t o n e a l s u r g e r y is a m a j o r cause of infertility, pain, bowel obstruction, and s u b s e q u e n t i n t r a o p e r a t i v e complications (1). T h e r e f o r e , a m e t h o d by which postsurgical a d h e s i o n f o r m a t i o n could be r e d u c e d or p r e v e n t e d would be of benefit. S t u d i e s h a v e i n d i c a t e d t h a t p l a c e m e n t of absorbable b a r r i e r s such as oxidized r e g e n e r a t e d cellulose (Interceed; J o h n s o n & J o h n s o n Medical I n c . , A r l i n g t o n , TX) and a composite film of carboxyReceived May 20, 1996; revised and accepted October 4, 1996. * Supported in part by a grant from the Ethicon Corporation, Somerville, New Jersey. t Livingston Reproductive Research Laboratory, University of Southern California School of Medicine. $ Reprint requests: Kathleen Rodgers, Ph.D., Livingston Reproductive Biology Laboratory, University of Southern California School of Medicine, 1321 North Mission Road, Los Angeles, California 90033 (FAX: 213-222-7038). § Ethicon, Inc. Vol. 67, No. 3, March 1997
m e t h y l cellulose a n d h y a l u r o n i c acid (Seprafilm; G e n z y m e Corporation, C a m b r i d g e , MA), or p e r m a n e n t b a r r i e r s like p o l y t e t r a f l u o r o e t h y l e n e (Preclude; W.L. Gore C o m p a n y , Flagstaff, AZ) b e t w e e n i n j u r y sites are effective in the r e d u c t i o n of a d h e s i o n formation (2). In the case of I n t e r c e e d , Seprafilm, or Preclude the s u r g e o n m u s t predict p o t e n t i a l sites of adhesion f o r m a t i o n to optimize b a r r i e r efficacy. T h e use of H y s k o n ( P h a r m a c i a , P i s c a t a w a y , NJ), a liquid a b s o r b a b l e b a r r i e r used to r e d u c e a d h e s i o n formation in clinical practice h a s s h o w n some u n d e s i r a b l e side effects r e s u l t i n g from the a c c u m u l a t i o n of i n t r a p e r i t o n e a l ascites due to its oncotic properties. In addition, studies h a v e indicated, t h a t i n t r a p e r i t o n e a l H y s k o n use is ineffective in a d h e s i o n r e d u c t i o n (3). R e c e n t studies d e m o n s t r a t e d t h a t h y a l u r o n i c acid r e d u c e d a d h e s i o n f o r m a t i o n in several situations, including h e a l i n g of s u b c u t a n e o u s , osseous, t e n d o n , ocular, a n d p e r i t o n e a l tissues ( 4 - 9 ) . H y a l u r o n i c acid Rodgers et al. Adhesion prevention by hyaluronic acid
553
is able to form viscous solutions t h a t appear to coat peritoneal surfaces and form a barrier to tissue apposition (10). In addition, hyaluronic acid modulates macrophage, lymphocyte, and polymorphonuclear neutrophil functions. The effect is either enhancement or suppression, depending upon the molecular weight and source of hyaluronic acid (11-18). These actions may be receptor-mediated because macrophages (i.e., the CD44 molecule on the macrophage) and fibroblasts express receptors for hyaluronic acid (19). In addition, hyaluronic acid may be involved in the wound repair process at several stages (2(~, 21). In this study, the effect of a single administration of hyaluronic acid from various sources and of different molecular weights and viscosities at the end of peritoneal surgery on intraperitoneal adhesion formation was examined in a standardized animal model. MATERIALS AND METHODS
Four hundred thirty-five rabbits underwent the surgical procedure listed below. All rabbits tolerated the surgery well. The National Institutes of Health guidelines for the Use of Animals in Research were followed and all procedures were approved by the University of Southern California Institutional Animal Care and Use Committee. Materials
Hyaluronic acid was obtained from Ethicon, Inc. ISomerville, NJ). Several preparations of hyaluronic acid were tested. B i l a t e r a l U t e r i n e H o r n A b r a s i o n and D e v a s c u l a r i z a t i o n Model
New Zealand white female rabbits of reproductive age, 2.4 to 2.7 kg, were assigned randomly to various treatment groups. The medicament, hyaluronic acid, was administered by a single IP injection (usually 15 mL) after surgery as described below. The surgeon was blinded to the group assignment of a given rabbit. All animals underwent a midline laparotomy under ketamine and xylazine anesthesia. Both uterine horns underwent two standardized surgical injuries: abrasion of the serosal surface with sterile gauze until punctate bleeding developed and ischemia of the u t e r i n e horn induced by removal of the collateral blood supply by digital pressure with gauze. This surgical model previously was shown to induce consistent adhesion formation (22). For most studies, the medicament was injected through the peritoneum at the end of surgery. For studies examining the effects of hyaluronic acid administered at a site remote to the surgical injury, a catheter was placed anteriorly between the liver and the dia554
Rodgers et al. Adhesion prevention by hyaluronic acid
phragm and the hyaluronic acid was administered just before suturing the abdominal muscles. After traumatization, the abdominal wound was closed in two layers using 3 - 0 monofilament suture. Seven days after surgery, the animals were killed by pentobarbital overdosage and a second laparotomy was performed for evaluation of adhesion formation. A qualitative grading system for evaluation of adhesion formation was performed by two blinded observers as follows: [1] 0+, no adhesions; [2] 0.5+, light, filmy adhesions involving only the bladder; [3] 1+, light, filmy adhesions slightly more extensive than 0.5+; [4] 1.5+, adhesions more extensive and more dense than 1+; [5] 2+, adhesions not separable, between uterine horn and bladder or bowel, but not both; [6] 2.5+, adhesions slightly more extensive than 2+; [7] 3+, adhesions more dense and more extensive than 2.5+, but some movement of the uterine horns is possible; or [8] 4+, severe adhesions, not separable, unable to move horns. This scoring system took into account all organs involved in adhesion formation to the primary organ, the extent of adhesion formation, and the tenacity of the adhesions formed. If a disagreement regarding a score arose, the highest score was assigned to the rabbit. Data Analysis and Statistics
The data were analyzed by one-way analysis of variance on the ranks. A P value < 0.05 was considered significant. RESULTS
Hyaluronic acid was tested for its ability to reduce adhesion formation after intraperitoneal trauma. The results using a relatively high-molecular-weight hyaluronic acid (approximately 2 x 106 d) are shown in Figure 1A. By rank order analysis, all hyaluronic acid solutions reduced the formation of adhesions. However, a solution of >-0.51% hyaluronic acid further reduced the extent and severity of adhesion formation. Above this concentration of hyaluronic acid, the efficacy of high-molecular-weight hyaluronic acid was not increased. At a concentration of 1% hyaluronic acid, the efficacy of the high-molecularweight hyaluronic acid was still evident, however, somewhat diminished (Table 1). Three intermediate-molecular-weight hyaluronic acid preparations also were tested in this model (Fig. 1B). When assessed by rank order analysis, all solutions reduced adhesion formation compared with controls. The highest molecular weight hyaluronic acid tested in this study (1.4 x 106 d) was the most efficacious in reducing adhesion formation. Two lower molecular weight hyaluronic acids (1.2 x 106 and 1.0 x 106 d) were tested at higher concentrations Fertility and Sterility ~
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second study examined a range of viscosities and found all to be efficacious with the 500 to 1,500 CPS formulation showing the greatest adhesion reduction as determined by rank order analysis of the data (Fig. 2B; Table 1). Various volumes of the low-viscosity, low-molecular-weight hyaluronic acid preparation were examined in an a t t e m p t to determine the m i n i m u m "dose" necessary to reduce adhesion formation (Fig. 3). As shown in Figure 3A, administration of 1 mL hyaluronic acid (1,000 CPS, 0.5% of 0.75 × l0 s d) effec-
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F i g u r e 1 Hyaluronic acid at various percent c o n c e n t r a t i o n s (0% hyaluronic acid-surgery only) w a s placed IP into r a b b i t s t h a t had u n d e r g o n e devascularization and a b r a s i o n of both u t e r i n e horns. Seven days later, the r a b b i t s were killed and the IP a d h e s i o n s were scored. CA}, The r e s u l t s from h i g h e r molecular weight hyaluronic acid (2 × 106 d; 15 mL} is shown. (B), The r e s u l t s of hyaluronic acid three different i n t e r m e d i a t e molecular w e i g h t s ( 1, 1.2, and 1.4 × 106 d; 15 mL) is shown.
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so t h a t all three hyaluronic acid preparations had equivalent viscosity. These formulations were highly efficacious at reducing adhesion formation when the mean rank was compared with control (Fig. 1A). However, if these two lower molecular weight hyaluronic acids were prepared at the same concentration as the 1.4 × l0 s d hyaluronic acid (and therefore a lower viscosity), they were not as efficacious (Table 1). Lastly, a lower molecular weight hyaluronic acid (0.75 × l0 s d) also was evaluated in this surgical model. An initial dose finding study indicated t h a t this low-molecular-weight hyaluronic acid was highly effective at preventing adhesions (Fig. 2A). A Vol. 67, No. 3, March 1997
R a n k A n a l y s i s of Adhesion Score*
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* The n u m b e r of r a b b i t s t h a t h a d a score of -<1.5 for each s t u d y is shown. In addition, the r e s u l t of the r a n k order a n a l y s i s of the scores is s h o w n . t Values are m e a n s _+ SEM. C o m p a r e d with controls. § These g r o u p s are significantly reduced c o m p a r e d with 0.36% hyaluronic acid. UThese g r o u p s are significantly reduced c o m p a r e d with 1.4 x 10 ~, 1.25%. ¶ T h e s e g r o u p s are significantly reduced c o m p a r e d with 1.1% i500 to 1500 CPS) hyaluronic acid. ** T h e s e g r o u p s are significantly reduced c o m p a r e d with 1 m L volume. R o d g e r s e t al.
Adhesion prevention by hyaluronic acid
555
morbidity and mortality of the postoperative patient. Increased awareness by surgeons of intraoperative techniques that decrease tissue t r a u m a to areas sensitive to adhesion formation and the use ofintraperitoneal antiadhesion adjuvants has lead to improved postoperative outcomes (1, 2). Intraperitoneal medic~aments, such as cortisone, and barrier therapies, such as Interceed, Preclude, and Seprafilm, have been shown to decrease adhesion formation in many surgical procedures. Previous studies in h u m a n s demonstrated t h a t hyaluronic acid was efficacious in the prevention of intraocular and tendon adhesions (4-7). In this study, the effect of hyaluronic
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B F i g u r e 2 Low-molecular-weight hyaluronic acid lapproximately 7.5 × 10 r' d; 15 mL) at various percent concentrations was tested for efficacy in the reduction of adhesion formation after abrasion and devascularization of both uterine horns in rabbits 10c~ hyaluronic acid-surgery only}. {A), The results of a pilot study where a small n u m b e r of hyaluronic acid viscosities were tested are presented. (B), This s a m e range ofviscosities with more intermediate viscosity points is shown. Seven days after surgical t r a u m a , the rabbits were killed and their adhesions were scored.
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tively reduced adhesion formation. However, lower volumes were not tested therefore a minimum volume for efficacy was not identified. When the lowmolecular-weight, low-viscosity hyaluronic acid was administered to the peritoneal cavity at a site remote from the abraded area, the medicament was still efficacious in the reduction of adhesion formation (Fig. 3B).
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F i g u r e 3 Low-viscosity (1,000 CPS), low-molecular-weight hyaluronic acid was administered at various volumes to rabbits t h a t had undergone devascularization and abrasion of both uterine horns. The results of a d m i n i s t r a t i o n of the formulation to the site of surgery (A) or at a site remote to the surgical t r a u m a (between the d i a p h r a g m and liver; (B) are shown. Seven days later, the rabbits were killed and their adhesion scores were evaluated. C, surgery only.
Adhesion prevention by hyaluronic acid
Fertility and Sterility ~
DISCUSSION
556
R o d g e r s e t al.
acid of various molecular weights and viscosities on the formation of intraperitoneal adhesions was examined. Most of the formulations tested were efficacious in the reduction of adhesions formation. However, the best formulation tested was a low-viscosity, low-molecular-weight hyaluronic acid. The high- (2 × 106 d) and intermediate- 1 to 1.4 × 106 d) molecular-weight hyaluronic acids were most efficacious in reducing adhesion formation at relatively low viscosities. However, this pattern was reversed when the lower molecular weight hyaluronic acid was tested (molecular weight approximately 7.5 × 10 "~d) in t h a t the low-viscosity formulation was the most efficacious. In addition, this formulation reduced adhesion formation even when administered at low volumes (i.e., volumes close to the minimum volume necessary to coat the peritoneal cavity of a rabbit) or when delivered at sites of the peritoneal cavity remote to the surgical trauma. A recent study by Grainger et al. (23) showed t h a t viscous hyaluronic acid, 1% solution of 3.1 x 106 d molecular weight hyaluronic acid or 1.4% solution of 4.7 × 10 ~ d molecular weight hyaluronic acid, was not effective in the reduction of adhesion formation after bisection of rabbit ovaries. It should be noted t h a t this viscosity would be higher (based on molecular weight and concentration) t h a n those tested here. In addition, the volume ofhyaluronic acid used in the Grainger et al. (23) study was sufficient to coat the entire peritoneal surface of a 2.5 kg rabbit. Therefore, the "nontreated" ovary in the rabbit may, in fact, have been exposed to hyaluronic acid in the postoperative interval. In which case, both ovaries would be expected to have similar adhesion scores, which was the experimental observation reported by Grainger et al. (23). Our data confirm and extend Grainger et al.'s (23) findings in t h a t lower molecular weight hyaluronic acid preparations are more effective than the higher molecular weight and higher viscosity hyaluronic acid preparations tested here. Recent studies from this laboratory showed t h a t tolmetin in an hyaluronic acid carrier (low-molecular-weight, high-viscosity hyaluronic acid; 8,000 CPS) significantly reduced the number of recoverable red blood cells (at 6, 48, and 72 hours) and increased the number of white blood cells (at 12 hours) in the peritoneal cavity after surgery (24). F u r t h e r studies showed t h a t administration of tolmetin in saline at the end of surgery transiently increased the number of red blood cells in the peritoneal lavage fluid (25). Therefore, hyaluronic acid may be involved in a reduction in the number of recoverable red blood cells from the peritoneal cavity and may have a hemostatic effect. Because hyaluronic acid is a component of normal wound fluid, it is expected t h a t its administration Vol. 67, No. 3, March 1997
into the peritoneal cavity would have minimal toxicity. In addition, upon necropsy of the rabbits treated with hyaluronic acid, no excess fluid retention was noted and therefore administration of isosmotic hyaluronic acid should not lead to ascites formation. In summary, several formulations of hyaluronic acid were examined for their efficacy in the reduction of postsurgical adhesion formation. Higher molecular weight hyaluronic acids were efficacious when high-viscosity formulations were administered, suggesting t h a t these preparations of the molecule acted as a barrier. In contrast, lower molecular weight hyaluronic acid was most efficacious when prepared in low-viscosity formulations even at low t r e a t m e n t volumes. This preparation therefore m a y act as a barrier to repel the surfaces of the traumatized peritoneum. REFERENCES 1. Thm'nton M, diZerega GS. Using banfiers to prevent adhesions. Contemp Ob Gyn 1996;41:107-24. 2. diZerega GS. Contemporary adhesion prevention. Fertil Steril 1994;61:219-35. 3. diZerega GS, Campeau JD. Use ofinstillates to prevent intraperitoneal adhesions; Crystalloid and Dextran. Infert Reprod Med Clin 1994;5:463-78. 4. Thomas SC, Jones LC, Hungerford DS. Hyaluronic acid and its effects on postoperative adhesions in the rabbit flexor tendon. Clin Orthop Relat Res 1982;206:281-9. 5. Weiss C, Balazs EA, St. Onge R, Denlinger JL. Clinical studies of the intra-articular injection of Healon (sodium hyaluronatel in the treatment of osteoarthritis of human knees. Semin Arthritis Rheum 1981;VII Suppl:143-7. 6. Weiss C, Levy HJ, Delinger J, Suros JM, Weiss HE. The role of Na-hylan in reducing postsurgical tendon adhesions. Bull Hosp Jt Dis Orthrop Inst 1986;45:9-15. 7. Weiss C, Suros JM, Michalow A, Denlinger J, Moore M, Tejero W. The role of Na-hylan in reducing postsurgical tendon adhesions: Part 2. Bull Hosp Jt Dis Orthop Inst 1987;47: 31-9. 8. Urman B, Gomel V. Effect ofhyaluronic acid on postoperative intraperitoneal adhesion formation and reformation in the rat model. Fertil Steril 1991;56:568-70. 9. Urman B, Gomel V, Jetha N. Effect of hyaluronic acid on postoperative intraperitoneal adhesion formation in the rat model. Fertil Steril 1991;56:563-7. 10. Morris ER, Rees DA, Welsh EJ. Conformation and dynamic interactions in hyaluronate solution. J Mol Biol 1983;138: 383-6. 11. Darzynkiewiez A, Balazs EA. Effect of connective tissue intracellular matrix on lymphocyte stimulation. I. Suppression of lymphocyte stimulation by hyaluronie acid. Exp Cell Res 1971;66:113-7. 12. Balazs EA, Darzynkiewicz A. The effect of hyaluronic acid on fibroblasts, mononuelear phagocytes and lymphocytes. In: Kulonen E, Pikkarainen J, editors. Biology of the fibroblast. New York: 1973:237-49. 13. Pessae B, Defendi V. Cell aggregation: role of acid mueopolysaecharides. Science 1972; 172:898-903. 14. Hakansson L, Hallgren R, Venge P. Regulation ofgranulocyte function by hyaluronic acid in vitro and in vivo effects phagocytosis, locomotion and metabolism. J Clin Invest 1980;66: 298-304. R o d g e r s e t al. Adhesion prevention by hyaluronic acid
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15. Forrester V, Balazs EA. Inhibition of phagocytosis by high molecular weight hyaluronate. Endocrinology 1980;40:43542. 16. Shannon BT, Love SH, Myroik QN. Participation of hyaluronic acid in the macrophage disappearance reaction. Immunol Commun 1980;9:735-46. 17. Ahlgren T, Jarstrand C. Hyaluronic acid enhances phagocytosis ofhuman monocytes in vitro. J Clin Immunol 1984;4:24652. 18. Hakansson L, Venge P. The molecular basis of the hyaluronic acid-mediated stimulation of the granulocyte function. J Immunol 1987; 138:4347-51. 19. Green SJ, Tarone G, Underhill CB. Aggregation of nfacrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor. Exp Cell Res 1988;178:224-9. 20. Weigel PH, Fuller GM, LeBoeuf RD. A model for the role of hyaluronic acid and fibrin in the early events during the
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21. 22.
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24.
25.
inflammatotT response and wound healing. J Theor Biol 1986; 119:219-34. diZerega GS, Rodgers KE. Extracellular matrix proteins. In: The peritoneum. New York: Springer Verlag, 1992:145-70. Nishimura K, Nakamura RM, diZerega GS. Ibuprofen inhibition ofpostsurgical adhesion formation: a time dose response biochemical evaluation in rabbits. J Surg Res 1984;36:11524. Grainger DA, Meyer WR, DeCherney AH, Diamond MP. The use of hyaluronic acid to reduce postoperative adhesions. J Gynecol Surg 1991;7:97-103. Abe H, Rodgers KE, Campeau JD, GirDs W, Ellefson D, diZerega GS. The effect of intraperitoneal administration of sodium tolmetin-hyaluronic acid on the postsurgical cell infiltration in vivo. J Surg Res 1990;49:322-7. Rodgers KE, Ellefson DD, Girgis W, diZerega GS. Modulation of postsurgical cell infiltration and fibrinolytic activity by tolmetin in two species. J Surg Res 1994;56:314-25.
Fertility and Sterility ®
Vol. 67, No. 3, March 1997
Cop:~ight ~ 1997 American Society for Reproductive Medicine
Printed on acid-free paper in U. S. A.
Reduction of adhesion formation with hyaluronic acid after peritoneal surgery in rabbits*
Kathleen E. Rodgers, Ph.D.t$ Douglas B. Johns, Ph.D.§ Wefki Girgis, D.V.M.t
Joseph Campeau, M.S.t Gere S. diZerega, M.D.t
University of Southern California School of Medicine, Los Angeles, California, and Ethicon, Inc., Somerville, New Jersey
Objective: To examine the effect ofhyaluronic acid, a high-molecular-weight glucosaminoglycan found in the extracellular matrix, on the formation of adhesions, a major source of postoperative complications. Design: The ability of hyaluronic acid to reduce adhesion formation was evaluated using a standardized rabbit model. The material was administered IP at the end of surgery. Setting: University laboratory. Animal(s): New Zealand White female rabbits. I n t e r v e n t i o n ( s ) : Intraperitoneal administration of various formulations of hyaluronic acid at the end of surgery. Main O u t c o m e Measure(s): One week after surgery, a second laparotomy was performed and the extent of adhesion formation was determined. Result(s): Five separate molecular weight ranges ofhyaluronic acid representing eight viscosities between 1,000 and 12,000 centipoise (CPS) were shown to reduce adhesion formation in this model. All volumes, 1 to 30 mL, of hyaluronic acid tested reduced adhesion formation. In addition, the low-viscosity, low-molecular-weight hyaluronic acid significantly reduced adhesion formation when added to the trauma site or when injected at a site remote from the trauma area. Conclusion(s): This study showed that hyaluronic acid administered at the end of surgery reduced adhesion formation. Fertil Steril ® 1997;67:553-8 Key Words: Hyaluronic acid, adhesion, intraperitoneal, viscoelastic
A d h e s i o n f o r m a t i o n a f t e r p e r i t o n e a l s u r g e r y is a m a j o r cause of infertility, pain, bowel obstruction, and s u b s e q u e n t i n t r a o p e r a t i v e complications (1). T h e r e f o r e , a m e t h o d by which postsurgical a d h e s i o n f o r m a t i o n could be r e d u c e d or p r e v e n t e d would be of benefit. S t u d i e s h a v e i n d i c a t e d t h a t p l a c e m e n t of absorbable b a r r i e r s such as oxidized r e g e n e r a t e d cellulose (Interceed; J o h n s o n & J o h n s o n Medical I n c . , A r l i n g t o n , TX) and a composite film of carboxyReceived May 20, 1996; revised and accepted October 4, 1996. * Supported in part by a grant from the Ethicon Corporation, Somerville, New Jersey. t Livingston Reproductive Research Laboratory, University of Southern California School of Medicine. $ Reprint requests: Kathleen Rodgers, Ph.D., Livingston Reproductive Biology Laboratory, University of Southern California School of Medicine, 1321 North Mission Road, Los Angeles, California 90033 (FAX: 213-222-7038). § Ethicon, Inc. Vol. 67, No. 3, March 1997
m e t h y l cellulose a n d h y a l u r o n i c acid (Seprafilm; G e n z y m e Corporation, C a m b r i d g e , MA), or p e r m a n e n t b a r r i e r s like p o l y t e t r a f l u o r o e t h y l e n e (Preclude; W.L. Gore C o m p a n y , Flagstaff, AZ) b e t w e e n i n j u r y sites are effective in the r e d u c t i o n of a d h e s i o n formation (2). In the case of I n t e r c e e d , Seprafilm, or Preclude the s u r g e o n m u s t predict p o t e n t i a l sites of adhesion f o r m a t i o n to optimize b a r r i e r efficacy. T h e use of H y s k o n ( P h a r m a c i a , P i s c a t a w a y , NJ), a liquid a b s o r b a b l e b a r r i e r used to r e d u c e a d h e s i o n formation in clinical practice h a s s h o w n some u n d e s i r a b l e side effects r e s u l t i n g from the a c c u m u l a t i o n of i n t r a p e r i t o n e a l ascites due to its oncotic properties. In addition, studies h a v e indicated, t h a t i n t r a p e r i t o n e a l H y s k o n use is ineffective in a d h e s i o n r e d u c t i o n (3). R e c e n t studies d e m o n s t r a t e d t h a t h y a l u r o n i c acid r e d u c e d a d h e s i o n f o r m a t i o n in several situations, including h e a l i n g of s u b c u t a n e o u s , osseous, t e n d o n , ocular, a n d p e r i t o n e a l tissues ( 4 - 9 ) . H y a l u r o n i c acid Rodgers et al. Adhesion prevention by hyaluronic acid
553
is able to form viscous solutions t h a t appear to coat peritoneal surfaces and form a barrier to tissue apposition (10). In addition, hyaluronic acid modulates macrophage, lymphocyte, and polymorphonuclear neutrophil functions. The effect is either enhancement or suppression, depending upon the molecular weight and source of hyaluronic acid (11-18). These actions may be receptor-mediated because macrophages (i.e., the CD44 molecule on the macrophage) and fibroblasts express receptors for hyaluronic acid (19). In addition, hyaluronic acid may be involved in the wound repair process at several stages (2(~, 21). In this study, the effect of a single administration of hyaluronic acid from various sources and of different molecular weights and viscosities at the end of peritoneal surgery on intraperitoneal adhesion formation was examined in a standardized animal model. MATERIALS AND METHODS
Four hundred thirty-five rabbits underwent the surgical procedure listed below. All rabbits tolerated the surgery well. The National Institutes of Health guidelines for the Use of Animals in Research were followed and all procedures were approved by the University of Southern California Institutional Animal Care and Use Committee. Materials
Hyaluronic acid was obtained from Ethicon, Inc. ISomerville, NJ). Several preparations of hyaluronic acid were tested. B i l a t e r a l U t e r i n e H o r n A b r a s i o n and D e v a s c u l a r i z a t i o n Model
New Zealand white female rabbits of reproductive age, 2.4 to 2.7 kg, were assigned randomly to various treatment groups. The medicament, hyaluronic acid, was administered by a single IP injection (usually 15 mL) after surgery as described below. The surgeon was blinded to the group assignment of a given rabbit. All animals underwent a midline laparotomy under ketamine and xylazine anesthesia. Both uterine horns underwent two standardized surgical injuries: abrasion of the serosal surface with sterile gauze until punctate bleeding developed and ischemia of the u t e r i n e horn induced by removal of the collateral blood supply by digital pressure with gauze. This surgical model previously was shown to induce consistent adhesion formation (22). For most studies, the medicament was injected through the peritoneum at the end of surgery. For studies examining the effects of hyaluronic acid administered at a site remote to the surgical injury, a catheter was placed anteriorly between the liver and the dia554
Rodgers et al. Adhesion prevention by hyaluronic acid
phragm and the hyaluronic acid was administered just before suturing the abdominal muscles. After traumatization, the abdominal wound was closed in two layers using 3 - 0 monofilament suture. Seven days after surgery, the animals were killed by pentobarbital overdosage and a second laparotomy was performed for evaluation of adhesion formation. A qualitative grading system for evaluation of adhesion formation was performed by two blinded observers as follows: [1] 0+, no adhesions; [2] 0.5+, light, filmy adhesions involving only the bladder; [3] 1+, light, filmy adhesions slightly more extensive than 0.5+; [4] 1.5+, adhesions more extensive and more dense than 1+; [5] 2+, adhesions not separable, between uterine horn and bladder or bowel, but not both; [6] 2.5+, adhesions slightly more extensive than 2+; [7] 3+, adhesions more dense and more extensive than 2.5+, but some movement of the uterine horns is possible; or [8] 4+, severe adhesions, not separable, unable to move horns. This scoring system took into account all organs involved in adhesion formation to the primary organ, the extent of adhesion formation, and the tenacity of the adhesions formed. If a disagreement regarding a score arose, the highest score was assigned to the rabbit. Data Analysis and Statistics
The data were analyzed by one-way analysis of variance on the ranks. A P value < 0.05 was considered significant. RESULTS
Hyaluronic acid was tested for its ability to reduce adhesion formation after intraperitoneal trauma. The results using a relatively high-molecular-weight hyaluronic acid (approximately 2 x 106 d) are shown in Figure 1A. By rank order analysis, all hyaluronic acid solutions reduced the formation of adhesions. However, a solution of >-0.51% hyaluronic acid further reduced the extent and severity of adhesion formation. Above this concentration of hyaluronic acid, the efficacy of high-molecular-weight hyaluronic acid was not increased. At a concentration of 1% hyaluronic acid, the efficacy of the high-molecularweight hyaluronic acid was still evident, however, somewhat diminished (Table 1). Three intermediate-molecular-weight hyaluronic acid preparations also were tested in this model (Fig. 1B). When assessed by rank order analysis, all solutions reduced adhesion formation compared with controls. The highest molecular weight hyaluronic acid tested in this study (1.4 x 106 d) was the most efficacious in reducing adhesion formation. Two lower molecular weight hyaluronic acids (1.2 x 106 and 1.0 x 106 d) were tested at higher concentrations Fertility and Sterility ~
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second study examined a range of viscosities and found all to be efficacious with the 500 to 1,500 CPS formulation showing the greatest adhesion reduction as determined by rank order analysis of the data (Fig. 2B; Table 1). Various volumes of the low-viscosity, low-molecular-weight hyaluronic acid preparation were examined in an a t t e m p t to determine the m i n i m u m "dose" necessary to reduce adhesion formation (Fig. 3). As shown in Figure 3A, administration of 1 mL hyaluronic acid (1,000 CPS, 0.5% of 0.75 × l0 s d) effec-
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F i g u r e 1 Hyaluronic acid at various percent c o n c e n t r a t i o n s (0% hyaluronic acid-surgery only) w a s placed IP into r a b b i t s t h a t had u n d e r g o n e devascularization and a b r a s i o n of both u t e r i n e horns. Seven days later, the r a b b i t s were killed and the IP a d h e s i o n s were scored. CA}, The r e s u l t s from h i g h e r molecular weight hyaluronic acid (2 × 106 d; 15 mL} is shown. (B), The r e s u l t s of hyaluronic acid three different i n t e r m e d i a t e molecular w e i g h t s ( 1, 1.2, and 1.4 × 106 d; 15 mL) is shown.
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so t h a t all three hyaluronic acid preparations had equivalent viscosity. These formulations were highly efficacious at reducing adhesion formation when the mean rank was compared with control (Fig. 1A). However, if these two lower molecular weight hyaluronic acids were prepared at the same concentration as the 1.4 × l0 s d hyaluronic acid (and therefore a lower viscosity), they were not as efficacious (Table 1). Lastly, a lower molecular weight hyaluronic acid (0.75 × l0 s d) also was evaluated in this surgical model. An initial dose finding study indicated t h a t this low-molecular-weight hyaluronic acid was highly effective at preventing adhesions (Fig. 2A). A Vol. 67, No. 3, March 1997
R a n k A n a l y s i s of Adhesion Score*
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* The n u m b e r of r a b b i t s t h a t h a d a score of -<1.5 for each s t u d y is shown. In addition, the r e s u l t of the r a n k order a n a l y s i s of the scores is s h o w n . t Values are m e a n s _+ SEM. C o m p a r e d with controls. § These g r o u p s are significantly reduced c o m p a r e d with 0.36% hyaluronic acid. UThese g r o u p s are significantly reduced c o m p a r e d with 1.4 x 10 ~, 1.25%. ¶ T h e s e g r o u p s are significantly reduced c o m p a r e d with 1.1% i500 to 1500 CPS) hyaluronic acid. ** T h e s e g r o u p s are significantly reduced c o m p a r e d with 1 m L volume. R o d g e r s e t al.
Adhesion prevention by hyaluronic acid
555
morbidity and mortality of the postoperative patient. Increased awareness by surgeons of intraoperative techniques that decrease tissue t r a u m a to areas sensitive to adhesion formation and the use ofintraperitoneal antiadhesion adjuvants has lead to improved postoperative outcomes (1, 2). Intraperitoneal medic~aments, such as cortisone, and barrier therapies, such as Interceed, Preclude, and Seprafilm, have been shown to decrease adhesion formation in many surgical procedures. Previous studies in h u m a n s demonstrated t h a t hyaluronic acid was efficacious in the prevention of intraocular and tendon adhesions (4-7). In this study, the effect of hyaluronic
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B F i g u r e 2 Low-molecular-weight hyaluronic acid lapproximately 7.5 × 10 r' d; 15 mL) at various percent concentrations was tested for efficacy in the reduction of adhesion formation after abrasion and devascularization of both uterine horns in rabbits 10c~ hyaluronic acid-surgery only}. {A), The results of a pilot study where a small n u m b e r of hyaluronic acid viscosities were tested are presented. (B), This s a m e range ofviscosities with more intermediate viscosity points is shown. Seven days after surgical t r a u m a , the rabbits were killed and their adhesions were scored.
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tively reduced adhesion formation. However, lower volumes were not tested therefore a minimum volume for efficacy was not identified. When the lowmolecular-weight, low-viscosity hyaluronic acid was administered to the peritoneal cavity at a site remote from the abraded area, the medicament was still efficacious in the reduction of adhesion formation (Fig. 3B).
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F i g u r e 3 Low-viscosity (1,000 CPS), low-molecular-weight hyaluronic acid was administered at various volumes to rabbits t h a t had undergone devascularization and abrasion of both uterine horns. The results of a d m i n i s t r a t i o n of the formulation to the site of surgery (A) or at a site remote to the surgical t r a u m a (between the d i a p h r a g m and liver; (B) are shown. Seven days later, the rabbits were killed and their adhesion scores were evaluated. C, surgery only.
Adhesion prevention by hyaluronic acid
Fertility and Sterility ~
DISCUSSION
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R o d g e r s e t al.
acid of various molecular weights and viscosities on the formation of intraperitoneal adhesions was examined. Most of the formulations tested were efficacious in the reduction of adhesions formation. However, the best formulation tested was a low-viscosity, low-molecular-weight hyaluronic acid. The high- (2 × 106 d) and intermediate- 1 to 1.4 × 106 d) molecular-weight hyaluronic acids were most efficacious in reducing adhesion formation at relatively low viscosities. However, this pattern was reversed when the lower molecular weight hyaluronic acid was tested (molecular weight approximately 7.5 × 10 "~d) in t h a t the low-viscosity formulation was the most efficacious. In addition, this formulation reduced adhesion formation even when administered at low volumes (i.e., volumes close to the minimum volume necessary to coat the peritoneal cavity of a rabbit) or when delivered at sites of the peritoneal cavity remote to the surgical trauma. A recent study by Grainger et al. (23) showed t h a t viscous hyaluronic acid, 1% solution of 3.1 x 106 d molecular weight hyaluronic acid or 1.4% solution of 4.7 × 10 ~ d molecular weight hyaluronic acid, was not effective in the reduction of adhesion formation after bisection of rabbit ovaries. It should be noted t h a t this viscosity would be higher (based on molecular weight and concentration) t h a n those tested here. In addition, the volume ofhyaluronic acid used in the Grainger et al. (23) study was sufficient to coat the entire peritoneal surface of a 2.5 kg rabbit. Therefore, the "nontreated" ovary in the rabbit may, in fact, have been exposed to hyaluronic acid in the postoperative interval. In which case, both ovaries would be expected to have similar adhesion scores, which was the experimental observation reported by Grainger et al. (23). Our data confirm and extend Grainger et al.'s (23) findings in t h a t lower molecular weight hyaluronic acid preparations are more effective than the higher molecular weight and higher viscosity hyaluronic acid preparations tested here. Recent studies from this laboratory showed t h a t tolmetin in an hyaluronic acid carrier (low-molecular-weight, high-viscosity hyaluronic acid; 8,000 CPS) significantly reduced the number of recoverable red blood cells (at 6, 48, and 72 hours) and increased the number of white blood cells (at 12 hours) in the peritoneal cavity after surgery (24). F u r t h e r studies showed t h a t administration of tolmetin in saline at the end of surgery transiently increased the number of red blood cells in the peritoneal lavage fluid (25). Therefore, hyaluronic acid may be involved in a reduction in the number of recoverable red blood cells from the peritoneal cavity and may have a hemostatic effect. Because hyaluronic acid is a component of normal wound fluid, it is expected t h a t its administration Vol. 67, No. 3, March 1997
into the peritoneal cavity would have minimal toxicity. In addition, upon necropsy of the rabbits treated with hyaluronic acid, no excess fluid retention was noted and therefore administration of isosmotic hyaluronic acid should not lead to ascites formation. In summary, several formulations of hyaluronic acid were examined for their efficacy in the reduction of postsurgical adhesion formation. Higher molecular weight hyaluronic acids were efficacious when high-viscosity formulations were administered, suggesting t h a t these preparations of the molecule acted as a barrier. In contrast, lower molecular weight hyaluronic acid was most efficacious when prepared in low-viscosity formulations even at low t r e a t m e n t volumes. This preparation therefore m a y act as a barrier to repel the surfaces of the traumatized peritoneum. REFERENCES 1. Thm'nton M, diZerega GS. Using banfiers to prevent adhesions. Contemp Ob Gyn 1996;41:107-24. 2. diZerega GS. Contemporary adhesion prevention. Fertil Steril 1994;61:219-35. 3. diZerega GS, Campeau JD. Use ofinstillates to prevent intraperitoneal adhesions; Crystalloid and Dextran. Infert Reprod Med Clin 1994;5:463-78. 4. Thomas SC, Jones LC, Hungerford DS. Hyaluronic acid and its effects on postoperative adhesions in the rabbit flexor tendon. Clin Orthop Relat Res 1982;206:281-9. 5. Weiss C, Balazs EA, St. Onge R, Denlinger JL. Clinical studies of the intra-articular injection of Healon (sodium hyaluronatel in the treatment of osteoarthritis of human knees. Semin Arthritis Rheum 1981;VII Suppl:143-7. 6. Weiss C, Levy HJ, Delinger J, Suros JM, Weiss HE. The role of Na-hylan in reducing postsurgical tendon adhesions. Bull Hosp Jt Dis Orthrop Inst 1986;45:9-15. 7. Weiss C, Suros JM, Michalow A, Denlinger J, Moore M, Tejero W. The role of Na-hylan in reducing postsurgical tendon adhesions: Part 2. Bull Hosp Jt Dis Orthop Inst 1987;47: 31-9. 8. Urman B, Gomel V. Effect ofhyaluronic acid on postoperative intraperitoneal adhesion formation and reformation in the rat model. Fertil Steril 1991;56:568-70. 9. Urman B, Gomel V, Jetha N. Effect of hyaluronic acid on postoperative intraperitoneal adhesion formation in the rat model. Fertil Steril 1991;56:563-7. 10. Morris ER, Rees DA, Welsh EJ. Conformation and dynamic interactions in hyaluronate solution. J Mol Biol 1983;138: 383-6. 11. Darzynkiewiez A, Balazs EA. Effect of connective tissue intracellular matrix on lymphocyte stimulation. I. Suppression of lymphocyte stimulation by hyaluronie acid. Exp Cell Res 1971;66:113-7. 12. Balazs EA, Darzynkiewicz A. The effect of hyaluronic acid on fibroblasts, mononuelear phagocytes and lymphocytes. In: Kulonen E, Pikkarainen J, editors. Biology of the fibroblast. New York: 1973:237-49. 13. Pessae B, Defendi V. Cell aggregation: role of acid mueopolysaecharides. Science 1972; 172:898-903. 14. Hakansson L, Hallgren R, Venge P. Regulation ofgranulocyte function by hyaluronic acid in vitro and in vivo effects phagocytosis, locomotion and metabolism. J Clin Invest 1980;66: 298-304. R o d g e r s e t al. Adhesion prevention by hyaluronic acid
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15. Forrester V, Balazs EA. Inhibition of phagocytosis by high molecular weight hyaluronate. Endocrinology 1980;40:43542. 16. Shannon BT, Love SH, Myroik QN. Participation of hyaluronic acid in the macrophage disappearance reaction. Immunol Commun 1980;9:735-46. 17. Ahlgren T, Jarstrand C. Hyaluronic acid enhances phagocytosis ofhuman monocytes in vitro. J Clin Immunol 1984;4:24652. 18. Hakansson L, Venge P. The molecular basis of the hyaluronic acid-mediated stimulation of the granulocyte function. J Immunol 1987; 138:4347-51. 19. Green SJ, Tarone G, Underhill CB. Aggregation of nfacrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor. Exp Cell Res 1988;178:224-9. 20. Weigel PH, Fuller GM, LeBoeuf RD. A model for the role of hyaluronic acid and fibrin in the early events during the
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21. 22.
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inflammatotT response and wound healing. J Theor Biol 1986; 119:219-34. diZerega GS, Rodgers KE. Extracellular matrix proteins. In: The peritoneum. New York: Springer Verlag, 1992:145-70. Nishimura K, Nakamura RM, diZerega GS. Ibuprofen inhibition ofpostsurgical adhesion formation: a time dose response biochemical evaluation in rabbits. J Surg Res 1984;36:11524. Grainger DA, Meyer WR, DeCherney AH, Diamond MP. The use of hyaluronic acid to reduce postoperative adhesions. J Gynecol Surg 1991;7:97-103. Abe H, Rodgers KE, Campeau JD, GirDs W, Ellefson D, diZerega GS. The effect of intraperitoneal administration of sodium tolmetin-hyaluronic acid on the postsurgical cell infiltration in vivo. J Surg Res 1990;49:322-7. Rodgers KE, Ellefson DD, Girgis W, diZerega GS. Modulation of postsurgical cell infiltration and fibrinolytic activity by tolmetin in two species. J Surg Res 1994;56:314-25.
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