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A novel hyaluronan-based gel in laparoscopic adhesion prevention: preclinical evaluation in an animal model
FERTILITY AND STERILITY@ VOL. 69, NO. 2, FEBRUARY 1998
Coovriaht . _ 01998 American Societv for Reoroductive Medicine Publis’hed by ksevier Science Inc. Printed on acid-free paper in U.S.A.
A novel hyaluronan-based gel in laparoscopic adhesion prevention: preclinical evaluation in an animal model Pier Andrea De lace, M.D.,* Marco Stefanetti, M.D.,* Daniele Pressato,t Simonetta Piana, M.D., $ Massimo Don&t Alessandra Pavesio,t and Luciano Bovicelli, M.D. * S. Orsola Hospital, University of Bologna, Italy
Objective: To evaluate the effectiveness adhesions
in laparoscopic
Design:
A randomized
Setting:
A standardized
Animals: Sixty-four
of a crosslinked
hyaluronan
solution (ACP gel) in the prevention
of postsurgical
surgery. blinded study using a rabbit model in laparoscopic
surgery.
surgical trauma in the rabbit uterine horn to induce adhesion formation.
sexually matured female New Zealand
white rabbits weighing
2.5 to 3.0 kg and aged 3-4 months.
Intervention(s): After trauma, group 1 (n = 22) received no treatment, group 2 animals (n = 20) received oxidizedregenerated
cellulose (Interceed
[TC7]), in group 3 (n = 22) 5 mL of ACP gel were applied on the lesion.
Main Outcome Measure(s): Six weeks after laparoscopy, according
a laparotomy
was performed
and the adhesions
were scored
to Blauer’s scoring system.
Result(s): 66% of the untreated animals and 8.5% of the animals treated with Interceed presented with severe adhesions, whereas only 35% of the ACP gel treatment group had significant adhesions. The mean (? SEM) increased adhesion score was 2.24 2 0.26 in the untreated group, 2.45 ? 0.22 in the Interceed group, and was 1.25 2 0.28 in the ACP gel group. Received June 10,1997; revised and accepted October 1,1997. Supported by Fidia Advanced Biopolymers s.r.I., Abano Terme, Italy. Reprint requests: Pier Andrea De lace, M.D., Osoedale S. Orsola. Clinica Ostetrica e Ginecologica, Universitl di Bologna, via Massarenti 13, 40138 Boloona. ltalv (FAX: 39-51349774):
zx
* S. Orsola Hospital, Department of Obstetrics and Gynecology, S. Orsola Hospital, University of Bologna, Bologna, Italy. t Fidia Advanced Biopolymeta s.r.l., Abano Terme, Italy. $ Department of Pathology, S. Orsola Hospital, University of Bologna, Bologna, Italy. 001%0282/98/$19.00 PII SO01 5-0282(98)00496-2
318
Conclusion(s): This study revealed that ACP gel holds promise as a novel resorbable biomaterial for the reduction of postoperative adhesions after laparoscopic surgery. (Fertil Steril o 1998;69:318-23. 01998 by American Society for Reproductive Medicine.)
Key Words: Adhesion prevention,
animal model, cross-linked
hyaluronan,
laparoscopic
surgery
Postsurgical adhesions develop following abdominal and gynecological surgery as a consequence of abnormal wound healing. Major complications that arise from postoperative adhesions include intestinal obstruction, infertility and chronic pain. A wide variety of barrier substances has been tested to prevent adhesions after open abdominal surgery (1). but few have been evaluated after laparoscopic surgery (2). Barrier materials are interposed between adjacent surfaces to avoid direct contact, however, unsatisfactory results have often been reported (3). To obtain low tissue reaction and, consequently, better clinical results, biodegradable barriers are desirable.
(HA), a natural component of the extracellular matrix, the vitreous humor, and synovial fluid of the joint. Hyaluronic acid has been experimentally shown to reduce postoperative adhesion formation after abdomino-pelvic (4,5) and orthopedic surgery (6); the antiadhesive effects depending on the molecular weight as well as the concentration of the preparation (7). However, the results are variable because unmodified HA is subject to rapid degradation and is cleared from the site of administration within hours. Recently, HA modified with foreign molecules in the form of a resorbable gel has been reported to significantly reduce the incidence and severity of adhesions (8,9).
One particularly promising biopolymer effectively acts as a barrier is hyaluronic
A new class of hyaluronic acid derivatives, the auto-crosslinked polysaccharides (ACP) se-
that acid
ries, has been developed with the aim of increasing the viscosity and the residence time of the gel upon application (10). The ACP polymers are obtained through a crosslinking process that results in a condensation and therefore does not introduce foreign bridge molecules (11). These chemically autocrosslinked polysaccharides are inter- and intra-molecular esters of HA (molecular weight 200,000 D) in which a part of the carboxyl group is esterified with hydroxyl groups of the same and/or different molecules of the polysaccharide. The ACP gel is a highly viscous suspension in sterile, distilled water of auto-reticulate microparticles, with variable concentration between 30 and 60 mg/mL. This crosslinked derivative is a biocompatible, biodegradable, noninflammatory material with improved viscoelastic properties compared to unmodified HA solutions of the same molecular weight (12). ACP has high adhesivity and prolonged residence time (13). As no foreign molecules are used to create the crosslink, the gel upon degradation is de-esterified and liberates hyaluronic acid, a naturally occurring molecule that has a biological role (14) and is eliminated through a known metabolic pathway (15). The aim of the present study was to evaluate the effectiveness of a preparation of ACP gel with defined viscosity and concentration for adhesion prevention in laparoscopic surgery. Several options were available in terms of choosing the correct gel but the most viscous one appeared more clinically relevant (60 mg/mL). A rabbit uterine horn injury was used as a standardized and reproducible model of experimental adhesion induction (16, 17). The ACP gel was tested in comparison to an absorbable oxidized-regenerated cellulose antiadhesive barrier that is currently available in clinical practice for this indication (Interceed [TC7])(18, 19) and an untreated control group (sham operated).
MATERIALS AND METHODS ACP gel (Fidia Advanced Biopolymers s.r.l., Abano Terme, Italy) was prepared by hydration of the powder to obtain a concentration of 6%. The relative crosslinking level was 5%. The transparent gel had a viscosity of 450 Pa s-l, and was steam sterilized in 5 mL glass syringes. All surgical procedures described were conducted in compliance with the Good Laboratory Practice guidelines and under sterile controlled conditions in appropriate room for middle size animal surgery at the animal medicine and care unit. Animal care and surgery were performed under Ministry of Health regulation #116/92. Date of notification November 4, 1996. Sixty-four sexually matured female New Zealand white rabbits weighing 2.5 to 3.0 kg and aged 3-4 months were housed under controlled environmental conditions at the Fidia Research Laboratories Animal Care. At the time of surgery, animals were anesthetized with 50 mgkg ketamine hydrochloride (Ketavet, Gellini PharmaceuFERTILITY
& STERILITY@
tical, Aprilia, Italy) and 1.6 mg/kg xylazine hydrochloride (Rompun, Bayer AG, Leverkusen, Germany). Additionally 5 mg/kg enrofloxacin (Baytril, Bayer AG, Leverkusen, Germany) was administered by SCinjection before surgery. The rabbits were then placed in the supine position, shaved in the abdomino-pelvic area, prepped with iodine solution and ethan01 then draped in sterile fashion. A Verres needle (1.2 mm in diameter) was inserted in the abdominal cavity entering the abdominal wall in the midline for carbon dioxide gas insufflation using an automatic laparoinsufflator (Storz, Tuttlingen, Germany) (intrabdominal pressure 8-12 mm Hg, flow 3 L/mm). A 4.5 mm diameter trocar was inserted entering the abdominal wall at the same position. A 4 mm diameter, 18 cm length, arthroscope (Storz) was inserted in the cavity through the trocar with the aid of a Xenon 300 W light source. All surgical procedures have been performed by means of an endoscopic microcamera (Mono CCD Endocam, Storz). After inspection of the abdomen, laparoscopic scissors and atraumatic forceps (5 mm in diameter) were inserted, without trocar, through two lateral incisions. A standardized injury was induced according to the following steps: [l] denuding a 2 cm2 area of the right uterine mesometrium; [2] crushing the medial right uterine horn for 30 seconds with forceps; [3] making a l-cm incision in the distal right uterine horn; [4] denuding a 5 cm2 area of the peritoneum of the abdominal wall in front of the previous lesions. Accurate hemostasis was obtained by monopolar electrocoagulation. The animals were randomized into three groups: 22 animals received no treatment (group 1, untreated control); 20 animals were treated with oxidized-regenerated cellulose (group 2): the material was carefully folded before use and inserted through a lateral abdominal incision. Interceed was unfolded inside the peritoneal cavity by means of forceps and applied on the lesion (Fig. 1A). In group 3, (22 animals) 5 mL of ACP gel were injected by means of a glass syringe with a 18-gauge needle inserted in the abdomen. The gel was uniformly distributed on all the injured areas (Fig. 2A). At the end of surgery all rabbits were treated with 0.8 mg/kg nefopam (Nefam, Farma-Biagini, Naples, Italy). Six weeks later, animals were euthanized by CO, asphyxiation, a laparotomy was performed, and the adhesion grade was blindly evaluated according to Blauer’s scoring system (20): 0 = no adhesions; 1 = thin or narrow, easily separable adhesions; 2 = thick adhesions, limited to one area; 3 = thick and widespread adhesions; 4 = thick and widespread adhesions, plus adhesions of viscera to anterior and/or posterior abdominal wall. A biopsy of adhesions was performed in some animals where moderate and severe adhesions were present. Tissue was removed and fixed in 10% formalin, and stained with hematoxylin and eosin. Histologic analysis evaluated: flogosis, granulation tissue, giant cell 319
Oxidized-regenerated cellulose treatment in laparoscopic surgery. In (A) the barrier has been unfolded in the peritoneal cavity and has been applied to the injured areas. In (B), the animal was sacrificed after 6 weeks, and adhesions between organs were noted.
reaction, fibrosis, fat necrosis, macrophage granulomatosis, rated on a 0 to 3 scale.
presence
and
The Kruskall-Wallis nonparametric test was used to compare the treatment and control groups. P < 0.05 was defined as statistically significant. Data are expressed as means t SEM.
RESULTS Three animals died from 4 to 5 days after surgery (two from the ACP group and one from the untreated group). Two rabbits presented intestinal stenosis due to electrical injury. The third rabbit died of intestinal hernia through the laparoscopic incision of the abdominal wall resulting in bowel necrosis. During surgical procedures, ACP gel was easily applied and adhered to the surfaces also when vertically positioned. High resistance due to the viscosity was encountered whilst applying the gel through a 18-gauge needle; all injured areas were completely covered, although the gel layer did not appear uniform. At the time of second-look
320
De Iaco et al.
laparotomy ACP was completely absorbed whereas nants of oxidized-regenerated cellulose were present.
rem-
The ACP treatment group showed the lowest incidence of postsurgical adhesions as compared to the oxidized-regenerated cellulose and untreated control groups (Fig. lB, Fig. 2B). The adhesion scores were 1.25 + 0.28,2.45 + 0.22 and 2.24 2 0.26 in the ACP, the oxidized-regenerated cellulose, and the untreated groups, respectively (Table 1). Statistically significant differences were noted between the ACP treated and the untreated control groups (P = 0.0058), and between the ACP and the oxidized-regenerated cellulose groups (P = 0.003), but not between the oxidized-regenerated cellulose treated and the untreated control groups (P = 0.1712). Clinically significant high grade adhesions (score ~2) were noted in 35% of the ACP-treated, in 66% of the control and in 85% of the oxidized-regenerated-cellulose-treated animals. No adhesions were observed in the left uterine horn where no trauma had been induced. The histologic findings of major adhesions showed no significant difference among the groups with respect to flo-
Hyaluronan derivative and adhesion prevention
Vol. 69, No. 2, February 1998
Autocrosslinked polysaccaride treatment in laparoscopic surgery. In (A) the gel has been uniformly applied on the injured areas. The material is adherent to all surfaces and is not cleared out after injection. In (6) the animal was sacrificed after 6 weeks, and no adhesions were noted.
gosis, granulation tissue, fat necrosis, and giant cells. The ACP treated group showed the lowest score of fibrosis, and significant differences were noted between the ACP gel treated group and the oxidized-regeneratedcellulose-treated group (P = 0.031). The analysis of macrophage presence showed differences between the untreated and the ACP treated groups vs. the oxidized-regenerated-cellulosetreated group (P = 0.014) (Table 2). Total absence of granulomatosis was found in all treatments.
sults are encouraging but often conflicting. Barrier methods are gaining interest because of their efficacy and absence of serious side effects. Their use is based upon the concept of covering the traumatized intrabdominal areas, thereby preventing direct apposition of surfaces and avoiding fibrin bridge formation between adjacent organs. Recently the use of highly viscous solutions or hydrogels such as carboxymethylcellulose (21), dextran (22), and hyaluronic acid (7) instilled after surgical procedures has been suggested as antiadhesive barrier methods.
DISCUSSION A large number of postsurgical adhesion methods have been suggested in the literature;
To obtain significant results, a modification of the standardized animal model proposed by De Iaco et al. (23) was used to ensure a high incidence of severe adhesion forma-
prevention clinical re-
Adhesion score and percentage of animals with adhesion score 22 in each treatment group. Score Treatment Control (untreated) Oxidized cellulose ACP Eel
No. of rabbits
0
1
2
3
4
Mean t SEM
21 20 20
1
6 3 6
4 9 3
I 4 3
3 4 1
2.24 2 0.26 2.45 2 0.22 1.25 t 0.28*
7
* P < 0.05 versus control group and versus oxidized cellulose,
FERTILITY & STERILITY@
non-parametric
Kruskall-Wallis
Incidence
of rabbits with score 22 66% 85% 35%
test.
321
Histologic features of biopsies from severe adhesions.
0
1
2
3
Mean 5 SEM
5 I 5
5 9 5
1 0 0
0 0
0.64 2 0.2 0.56 ? 0.13 0.73 2 0.21
6 13 5
5 2 5
0 0 0
0
group
1 1
0.45 + 0.16 0.31 ? 0.20 0.73 2 0.27
4 4 3
3 3 6
0
group
3 1
4 6 1
1.36 2 0.41 1.69 2 0.31 1.00 2 0.27
8 8
3 5 1
0
0
1 0
2 0
0.27 ? 0.14 0.81 2 0.26 0.09 + 0.09*
1 1 2
0
12 8
1 0
3 2 1
0.91 2 0.41 0.56 ” 0.27 0.45 * 0.28
9 6 9
2 3 2
0
0
5 0
2 0
0.18 ? 0.12 1.19 ? 0.28 0.18 + 0.12*
Histologic feature Flogosis Untreated group Oxidized cellulose ACP gel group Granulation tissue Untreated group Oxidized cellulose ACP gel group Giant cells Untreated group Oxidized cellulose ACP gel group Fibrosis Untreated group Oxidized cellulose ACP gel group Fat necrosis Untreated group Oxidized cellulose ACP gel group Macrophages Untreated group Oxidized cellulose ACP gel group
group
group
10 7
group
group
1
* P < 0.05 versus oxidized-regenerated cellulose, nonparametric Kruskall-Wallis test.
tion: the additional injury on the abdominal wall previously described, increased the incidence and severity of adhesions with the adjacent uterine horn. The same surgical procedure has been applied in all animals, and the early postoperative deaths should be ascribed to surgical complications; however, animals that completed the study were equally distributed in the three groups. The hyaluronic acid derivative ACP gel is a new biomaterial; it is highly viscous and therefore conformable to the uterine and abdomino-pelvic surfaces, and it remains adherent to the tissue, even in a vertical position, for a suitable period of time before degradation. The objective of this experimental preclinical study has been to demonstrate the efficacy of ACP gel in the prevention of postsurgical adhesions after laparoscopic surgery. The results of this study showed that ACP gel used postsurgically significantly reduced adhesion formation compared to an oxidized regenerated cellulose and to no postsurgical treatment. Thirty-five percent of rabbits in the ACP gel treated group presented no adhesions, while less than 5% in the control group and no rabbit in the oxidizedregenerated cellulose group did not develop postsurgical adhesions. Adhesions in the ACP gel treated animals were generally filmy and avascular, being severe only in 35% of
322
De Iaco et al.
Hyaluronan
derivative
cases. Control rabbits showed thick and widespread adhesions in 66% of cases. Oxidized regenerated cellulose did not prevent adhesion formation in 85% of cases in this animal model. These data confirm conflicting results of the efficacy of oxidized regenerated cellulose in animal models (8,24,25). Histologic analysis revealed ACP gel did not induce a specific inflammatory reaction, and caused lower fibrotic response compared to the untreated and oxidized-regenerated-cellulose-treated group. The efficacy of the hyaluronan based gel may be due to several features of the material, mainly high tolerability and prolonged residence time during the wound healing process. Because of the high viscosity and adhesivity of ACP gel, it is possible to easily cover all vertical lesions that are frequently found during laparoscopic operations. Some difficulties were encountered while applying the gel through a l&gauge needle because of the high resistance; consequently a more appropriate applicator should be developed before clinical trials.
Acknowledgments: The authors thank Zaccanti s.r.1. (Bologna, Italy) and Mrs. Chiara Bagnoli for useful technical support, Dr. Francesca Patamello
and adhesion prevention
Vol. 69, No. 2, February
1998
for assistance in statistical analysis, and Dr. Valerie Colletta for linguistic assistance.
References 1. Saravelos HG, Le TC. Physical barriers in adhesion prevention. J Reprod Med 1996;41:42-51. 2. Diamond MP, Hershlag A. Adhesion formation and re-formation. In: di Zerega GS, Diamond MP, Malinak R, Linsky CB, eds. Treatment of postsurgical adhesions. New York: Wiley-Liss Inc., 1990:27-37. 3. Haney AF, Helsa .I, Hurst BS, Kettel LM, Murphy AA, Rock J, et al. Exuanded oolvtetrafluoroethvlene (Gore-Tex Sureical Membrane) is superior toXox&zed regenerited cellulose (Interceid TC7) in prevkntina adhesions. Fertil Steril 1995:63:1021-6. 4. U&an B, Gomel V, Jeha N. Effect of hyaluronic acid on postoperative intraperitoneal adhesion formation in the rat model. Fertil Steril 1991; 56:563-7. 5. Grainger DA, Meyer WR, De Chemey AH, Diamond MP. The use of hyaluronic acid polymers to reduce postoperative adhesions. J Gynecol slug 1991;7:97-101. 6. Hagberg L, Gerdin B. Sodium hyahrronate as an adjunct in adhesion prevention after flexor tendon surgery in rabbits. .I Hand Surg 1992; 17A:935-41. I. Bums JW, Skinner K, Colt J, Sheidlin A, Bronson R, Yaacobi Y, et al. Prevention of tissue injury and postsurgical adhesions by precoating tissues with hyaluronic acid solutions. .J Surg Res 1995;59:644-52. 8. Hill-West JL, Chowdhury SM, Dunn RC, Hubbel JA. Efficacy of a resorbable hydrogel barrier, oxidized regenerated cellulose, and hyaluranic acid in the prevention of ovarian adhesions in a rabbit model. Fertil Steril 1994;62:630-4. 9. Bums JW, Skinner K, Colt J, Burgess L, Rose R, Diamond MP. A hyaluronate based gel for the prevention of postsurgical adhesions: evaluation in two animal species. Fertil Steril 1996;66:814-21. 10. Balazs EA, Leshchiner EA. Hyaluronan, its crosslinked derivative, Hylan, and their medical applications. In: Inagaki H, Phillips GO, eds. Proceedings of the Nisshinbo International Conference on cellulosics utilization in the near future. New York: Elsevier Applied Science, 1989:233-41. 11. Benedetti L, Bellini D, Renier D, O’Regan M. Chemical modification of hyaluronan. In: Williams DF, ed. Novel biomaterials based on hyaluronic acid and its derivatives. Liverpool: Williams, 1994: 20-9.
FERTILITY
& STERILITY@
12. Balazs EA, Bland PA, Denlinger JL, Goldman AI, Larsen NE, Leshchiner EA, et al. Matrix engineering. Blood Coag Fibrin 1991;2:173-8. 13. Mensitieri M, Ambrosio L, Nicolais L, Bellini D, O’Regan M. Viscoelastic orooerties modulation of a novel autocrosslinked hvaluronic acid polymei. J Mater Sci Mater med 1996;7:695-8. 14. King SR, Hickerson WL, Proctor KG. Beneficial actions of exogenous hyaluronic acid on wound healing. Surgery 1991;109:76-84. 15. Fraser JRE. Laurent TC, Engstriim-Laurent A, Laurent UGB. Elimination of hyaluronic acid fromthe blood stream in the human. Clin Exp Pharmacol Physiol 1984;11:17-25. 16. Diamond MP, De Chemey AH, Linsky CB, Cunningham T, Constantine B. Adhesion re-formation in the rabbit uterine horn model: reduction with carboxymethylcelluIose. Int J Fertil 1988;33:372-5. 17. Sawhney AS, Pathak CP, Van Rensburg JJ, Dunn RC, Hubbell JA. Optimization of photopolymerized bioerodible hydrogel properties for adhesion prevention. J Biomed Mater Res 1994;28:831-8. 18. Saravelos-H, Le TC. Post-operative adhesions after laparoscopic electrosurgical treatment for polycystic ovarian syndrome with the application of Interceedm to one ovary: a prospective randomized controlled study. Human Reprod 1996;11:992-7. 19. Li TC, Cooke ID. The value of an absorbable adhesion barrier, Interceeda, in the prevention of adhesion reformation following microsurgical adhesiolysis. Br J Obstet Gynecol 1994;101:335-9. 20. Blauer KL, Collins RL. The effect of intraperitoneal progesterone on postoperative adhesion formation in rabbits. Fertil Steril 1988; 49: 144-9. 21. Diamond MP, De Chemey AH, Linsky CB, Cunningham T, Constantine B. Adhesion reformation in the rabbit uterine horn model: reduction with carboxymethylcellulose. Int J Fert 1988;33:372-5. 22. Bouldoris 0, Autret M, Paris FX, Karaitianos I, Loisance D, HenrySchet J. Effect synergique du Dextran 70 a 32% et dun antibiotique dans la prevention des adherences p&itoneales. J Chir 1992; 129:160-4. 23. De Iaco PA, Costa A, Mazzoleni G, Pasquinelli G, Bassein L, Marabini A. Fibrin sealant in laparoscopic adhesion prevention in the rabbit uterine horn model. Fertil Steril 1994;62:400-4. 24. Best CL, Rittenhouse D, Sueldo CE. A comparison of TC7 and 32% dextran 70 for prevention of postoperative adhesions in hamsters. Obstet Gynecol 1991;78:858-60. 25. Pagidas K, Tulandi T. Effects of Ringer’s lactate, Interceeda (TC7) and Gore-Texm surgical membrane on postsurgical adhesion formation. Fertil Steril 1992:57:199-201.
323
Coovriaht . _ 01998 American Societv for Reoroductive Medicine Publis’hed by ksevier Science Inc. Printed on acid-free paper in U.S.A.
A novel hyaluronan-based gel in laparoscopic adhesion prevention: preclinical evaluation in an animal model Pier Andrea De lace, M.D.,* Marco Stefanetti, M.D.,* Daniele Pressato,t Simonetta Piana, M.D., $ Massimo Don&t Alessandra Pavesio,t and Luciano Bovicelli, M.D. * S. Orsola Hospital, University of Bologna, Italy
Objective: To evaluate the effectiveness adhesions
in laparoscopic
Design:
A randomized
Setting:
A standardized
Animals: Sixty-four
of a crosslinked
hyaluronan
solution (ACP gel) in the prevention
of postsurgical
surgery. blinded study using a rabbit model in laparoscopic
surgery.
surgical trauma in the rabbit uterine horn to induce adhesion formation.
sexually matured female New Zealand
white rabbits weighing
2.5 to 3.0 kg and aged 3-4 months.
Intervention(s): After trauma, group 1 (n = 22) received no treatment, group 2 animals (n = 20) received oxidizedregenerated
cellulose (Interceed
[TC7]), in group 3 (n = 22) 5 mL of ACP gel were applied on the lesion.
Main Outcome Measure(s): Six weeks after laparoscopy, according
a laparotomy
was performed
and the adhesions
were scored
to Blauer’s scoring system.
Result(s): 66% of the untreated animals and 8.5% of the animals treated with Interceed presented with severe adhesions, whereas only 35% of the ACP gel treatment group had significant adhesions. The mean (? SEM) increased adhesion score was 2.24 2 0.26 in the untreated group, 2.45 ? 0.22 in the Interceed group, and was 1.25 2 0.28 in the ACP gel group. Received June 10,1997; revised and accepted October 1,1997. Supported by Fidia Advanced Biopolymers s.r.I., Abano Terme, Italy. Reprint requests: Pier Andrea De lace, M.D., Osoedale S. Orsola. Clinica Ostetrica e Ginecologica, Universitl di Bologna, via Massarenti 13, 40138 Boloona. ltalv (FAX: 39-51349774):
zx
* S. Orsola Hospital, Department of Obstetrics and Gynecology, S. Orsola Hospital, University of Bologna, Bologna, Italy. t Fidia Advanced Biopolymeta s.r.l., Abano Terme, Italy. $ Department of Pathology, S. Orsola Hospital, University of Bologna, Bologna, Italy. 001%0282/98/$19.00 PII SO01 5-0282(98)00496-2
318
Conclusion(s): This study revealed that ACP gel holds promise as a novel resorbable biomaterial for the reduction of postoperative adhesions after laparoscopic surgery. (Fertil Steril o 1998;69:318-23. 01998 by American Society for Reproductive Medicine.)
Key Words: Adhesion prevention,
animal model, cross-linked
hyaluronan,
laparoscopic
surgery
Postsurgical adhesions develop following abdominal and gynecological surgery as a consequence of abnormal wound healing. Major complications that arise from postoperative adhesions include intestinal obstruction, infertility and chronic pain. A wide variety of barrier substances has been tested to prevent adhesions after open abdominal surgery (1). but few have been evaluated after laparoscopic surgery (2). Barrier materials are interposed between adjacent surfaces to avoid direct contact, however, unsatisfactory results have often been reported (3). To obtain low tissue reaction and, consequently, better clinical results, biodegradable barriers are desirable.
(HA), a natural component of the extracellular matrix, the vitreous humor, and synovial fluid of the joint. Hyaluronic acid has been experimentally shown to reduce postoperative adhesion formation after abdomino-pelvic (4,5) and orthopedic surgery (6); the antiadhesive effects depending on the molecular weight as well as the concentration of the preparation (7). However, the results are variable because unmodified HA is subject to rapid degradation and is cleared from the site of administration within hours. Recently, HA modified with foreign molecules in the form of a resorbable gel has been reported to significantly reduce the incidence and severity of adhesions (8,9).
One particularly promising biopolymer effectively acts as a barrier is hyaluronic
A new class of hyaluronic acid derivatives, the auto-crosslinked polysaccharides (ACP) se-
that acid
ries, has been developed with the aim of increasing the viscosity and the residence time of the gel upon application (10). The ACP polymers are obtained through a crosslinking process that results in a condensation and therefore does not introduce foreign bridge molecules (11). These chemically autocrosslinked polysaccharides are inter- and intra-molecular esters of HA (molecular weight 200,000 D) in which a part of the carboxyl group is esterified with hydroxyl groups of the same and/or different molecules of the polysaccharide. The ACP gel is a highly viscous suspension in sterile, distilled water of auto-reticulate microparticles, with variable concentration between 30 and 60 mg/mL. This crosslinked derivative is a biocompatible, biodegradable, noninflammatory material with improved viscoelastic properties compared to unmodified HA solutions of the same molecular weight (12). ACP has high adhesivity and prolonged residence time (13). As no foreign molecules are used to create the crosslink, the gel upon degradation is de-esterified and liberates hyaluronic acid, a naturally occurring molecule that has a biological role (14) and is eliminated through a known metabolic pathway (15). The aim of the present study was to evaluate the effectiveness of a preparation of ACP gel with defined viscosity and concentration for adhesion prevention in laparoscopic surgery. Several options were available in terms of choosing the correct gel but the most viscous one appeared more clinically relevant (60 mg/mL). A rabbit uterine horn injury was used as a standardized and reproducible model of experimental adhesion induction (16, 17). The ACP gel was tested in comparison to an absorbable oxidized-regenerated cellulose antiadhesive barrier that is currently available in clinical practice for this indication (Interceed [TC7])(18, 19) and an untreated control group (sham operated).
MATERIALS AND METHODS ACP gel (Fidia Advanced Biopolymers s.r.l., Abano Terme, Italy) was prepared by hydration of the powder to obtain a concentration of 6%. The relative crosslinking level was 5%. The transparent gel had a viscosity of 450 Pa s-l, and was steam sterilized in 5 mL glass syringes. All surgical procedures described were conducted in compliance with the Good Laboratory Practice guidelines and under sterile controlled conditions in appropriate room for middle size animal surgery at the animal medicine and care unit. Animal care and surgery were performed under Ministry of Health regulation #116/92. Date of notification November 4, 1996. Sixty-four sexually matured female New Zealand white rabbits weighing 2.5 to 3.0 kg and aged 3-4 months were housed under controlled environmental conditions at the Fidia Research Laboratories Animal Care. At the time of surgery, animals were anesthetized with 50 mgkg ketamine hydrochloride (Ketavet, Gellini PharmaceuFERTILITY
& STERILITY@
tical, Aprilia, Italy) and 1.6 mg/kg xylazine hydrochloride (Rompun, Bayer AG, Leverkusen, Germany). Additionally 5 mg/kg enrofloxacin (Baytril, Bayer AG, Leverkusen, Germany) was administered by SCinjection before surgery. The rabbits were then placed in the supine position, shaved in the abdomino-pelvic area, prepped with iodine solution and ethan01 then draped in sterile fashion. A Verres needle (1.2 mm in diameter) was inserted in the abdominal cavity entering the abdominal wall in the midline for carbon dioxide gas insufflation using an automatic laparoinsufflator (Storz, Tuttlingen, Germany) (intrabdominal pressure 8-12 mm Hg, flow 3 L/mm). A 4.5 mm diameter trocar was inserted entering the abdominal wall at the same position. A 4 mm diameter, 18 cm length, arthroscope (Storz) was inserted in the cavity through the trocar with the aid of a Xenon 300 W light source. All surgical procedures have been performed by means of an endoscopic microcamera (Mono CCD Endocam, Storz). After inspection of the abdomen, laparoscopic scissors and atraumatic forceps (5 mm in diameter) were inserted, without trocar, through two lateral incisions. A standardized injury was induced according to the following steps: [l] denuding a 2 cm2 area of the right uterine mesometrium; [2] crushing the medial right uterine horn for 30 seconds with forceps; [3] making a l-cm incision in the distal right uterine horn; [4] denuding a 5 cm2 area of the peritoneum of the abdominal wall in front of the previous lesions. Accurate hemostasis was obtained by monopolar electrocoagulation. The animals were randomized into three groups: 22 animals received no treatment (group 1, untreated control); 20 animals were treated with oxidized-regenerated cellulose (group 2): the material was carefully folded before use and inserted through a lateral abdominal incision. Interceed was unfolded inside the peritoneal cavity by means of forceps and applied on the lesion (Fig. 1A). In group 3, (22 animals) 5 mL of ACP gel were injected by means of a glass syringe with a 18-gauge needle inserted in the abdomen. The gel was uniformly distributed on all the injured areas (Fig. 2A). At the end of surgery all rabbits were treated with 0.8 mg/kg nefopam (Nefam, Farma-Biagini, Naples, Italy). Six weeks later, animals were euthanized by CO, asphyxiation, a laparotomy was performed, and the adhesion grade was blindly evaluated according to Blauer’s scoring system (20): 0 = no adhesions; 1 = thin or narrow, easily separable adhesions; 2 = thick adhesions, limited to one area; 3 = thick and widespread adhesions; 4 = thick and widespread adhesions, plus adhesions of viscera to anterior and/or posterior abdominal wall. A biopsy of adhesions was performed in some animals where moderate and severe adhesions were present. Tissue was removed and fixed in 10% formalin, and stained with hematoxylin and eosin. Histologic analysis evaluated: flogosis, granulation tissue, giant cell 319
Oxidized-regenerated cellulose treatment in laparoscopic surgery. In (A) the barrier has been unfolded in the peritoneal cavity and has been applied to the injured areas. In (B), the animal was sacrificed after 6 weeks, and adhesions between organs were noted.
reaction, fibrosis, fat necrosis, macrophage granulomatosis, rated on a 0 to 3 scale.
presence
and
The Kruskall-Wallis nonparametric test was used to compare the treatment and control groups. P < 0.05 was defined as statistically significant. Data are expressed as means t SEM.
RESULTS Three animals died from 4 to 5 days after surgery (two from the ACP group and one from the untreated group). Two rabbits presented intestinal stenosis due to electrical injury. The third rabbit died of intestinal hernia through the laparoscopic incision of the abdominal wall resulting in bowel necrosis. During surgical procedures, ACP gel was easily applied and adhered to the surfaces also when vertically positioned. High resistance due to the viscosity was encountered whilst applying the gel through a 18-gauge needle; all injured areas were completely covered, although the gel layer did not appear uniform. At the time of second-look
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De Iaco et al.
laparotomy ACP was completely absorbed whereas nants of oxidized-regenerated cellulose were present.
rem-
The ACP treatment group showed the lowest incidence of postsurgical adhesions as compared to the oxidized-regenerated cellulose and untreated control groups (Fig. lB, Fig. 2B). The adhesion scores were 1.25 + 0.28,2.45 + 0.22 and 2.24 2 0.26 in the ACP, the oxidized-regenerated cellulose, and the untreated groups, respectively (Table 1). Statistically significant differences were noted between the ACP treated and the untreated control groups (P = 0.0058), and between the ACP and the oxidized-regenerated cellulose groups (P = 0.003), but not between the oxidized-regenerated cellulose treated and the untreated control groups (P = 0.1712). Clinically significant high grade adhesions (score ~2) were noted in 35% of the ACP-treated, in 66% of the control and in 85% of the oxidized-regenerated-cellulose-treated animals. No adhesions were observed in the left uterine horn where no trauma had been induced. The histologic findings of major adhesions showed no significant difference among the groups with respect to flo-
Hyaluronan derivative and adhesion prevention
Vol. 69, No. 2, February 1998
Autocrosslinked polysaccaride treatment in laparoscopic surgery. In (A) the gel has been uniformly applied on the injured areas. The material is adherent to all surfaces and is not cleared out after injection. In (6) the animal was sacrificed after 6 weeks, and no adhesions were noted.
gosis, granulation tissue, fat necrosis, and giant cells. The ACP treated group showed the lowest score of fibrosis, and significant differences were noted between the ACP gel treated group and the oxidized-regeneratedcellulose-treated group (P = 0.031). The analysis of macrophage presence showed differences between the untreated and the ACP treated groups vs. the oxidized-regenerated-cellulosetreated group (P = 0.014) (Table 2). Total absence of granulomatosis was found in all treatments.
sults are encouraging but often conflicting. Barrier methods are gaining interest because of their efficacy and absence of serious side effects. Their use is based upon the concept of covering the traumatized intrabdominal areas, thereby preventing direct apposition of surfaces and avoiding fibrin bridge formation between adjacent organs. Recently the use of highly viscous solutions or hydrogels such as carboxymethylcellulose (21), dextran (22), and hyaluronic acid (7) instilled after surgical procedures has been suggested as antiadhesive barrier methods.
DISCUSSION A large number of postsurgical adhesion methods have been suggested in the literature;
To obtain significant results, a modification of the standardized animal model proposed by De Iaco et al. (23) was used to ensure a high incidence of severe adhesion forma-
prevention clinical re-
Adhesion score and percentage of animals with adhesion score 22 in each treatment group. Score Treatment Control (untreated) Oxidized cellulose ACP Eel
No. of rabbits
0
1
2
3
4
Mean t SEM
21 20 20
1
6 3 6
4 9 3
I 4 3
3 4 1
2.24 2 0.26 2.45 2 0.22 1.25 t 0.28*
7
* P < 0.05 versus control group and versus oxidized cellulose,
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non-parametric
Kruskall-Wallis
Incidence
of rabbits with score 22 66% 85% 35%
test.
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Histologic features of biopsies from severe adhesions.
0
1
2
3
Mean 5 SEM
5 I 5
5 9 5
1 0 0
0 0
0.64 2 0.2 0.56 ? 0.13 0.73 2 0.21
6 13 5
5 2 5
0 0 0
0
group
1 1
0.45 + 0.16 0.31 ? 0.20 0.73 2 0.27
4 4 3
3 3 6
0
group
3 1
4 6 1
1.36 2 0.41 1.69 2 0.31 1.00 2 0.27
8 8
3 5 1
0
0
1 0
2 0
0.27 ? 0.14 0.81 2 0.26 0.09 + 0.09*
1 1 2
0
12 8
1 0
3 2 1
0.91 2 0.41 0.56 ” 0.27 0.45 * 0.28
9 6 9
2 3 2
0
0
5 0
2 0
0.18 ? 0.12 1.19 ? 0.28 0.18 + 0.12*
Histologic feature Flogosis Untreated group Oxidized cellulose ACP gel group Granulation tissue Untreated group Oxidized cellulose ACP gel group Giant cells Untreated group Oxidized cellulose ACP gel group Fibrosis Untreated group Oxidized cellulose ACP gel group Fat necrosis Untreated group Oxidized cellulose ACP gel group Macrophages Untreated group Oxidized cellulose ACP gel group
group
group
10 7
group
group
1
* P < 0.05 versus oxidized-regenerated cellulose, nonparametric Kruskall-Wallis test.
tion: the additional injury on the abdominal wall previously described, increased the incidence and severity of adhesions with the adjacent uterine horn. The same surgical procedure has been applied in all animals, and the early postoperative deaths should be ascribed to surgical complications; however, animals that completed the study were equally distributed in the three groups. The hyaluronic acid derivative ACP gel is a new biomaterial; it is highly viscous and therefore conformable to the uterine and abdomino-pelvic surfaces, and it remains adherent to the tissue, even in a vertical position, for a suitable period of time before degradation. The objective of this experimental preclinical study has been to demonstrate the efficacy of ACP gel in the prevention of postsurgical adhesions after laparoscopic surgery. The results of this study showed that ACP gel used postsurgically significantly reduced adhesion formation compared to an oxidized regenerated cellulose and to no postsurgical treatment. Thirty-five percent of rabbits in the ACP gel treated group presented no adhesions, while less than 5% in the control group and no rabbit in the oxidizedregenerated cellulose group did not develop postsurgical adhesions. Adhesions in the ACP gel treated animals were generally filmy and avascular, being severe only in 35% of
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Hyaluronan
derivative
cases. Control rabbits showed thick and widespread adhesions in 66% of cases. Oxidized regenerated cellulose did not prevent adhesion formation in 85% of cases in this animal model. These data confirm conflicting results of the efficacy of oxidized regenerated cellulose in animal models (8,24,25). Histologic analysis revealed ACP gel did not induce a specific inflammatory reaction, and caused lower fibrotic response compared to the untreated and oxidized-regenerated-cellulose-treated group. The efficacy of the hyaluronan based gel may be due to several features of the material, mainly high tolerability and prolonged residence time during the wound healing process. Because of the high viscosity and adhesivity of ACP gel, it is possible to easily cover all vertical lesions that are frequently found during laparoscopic operations. Some difficulties were encountered while applying the gel through a l&gauge needle because of the high resistance; consequently a more appropriate applicator should be developed before clinical trials.
Acknowledgments: The authors thank Zaccanti s.r.1. (Bologna, Italy) and Mrs. Chiara Bagnoli for useful technical support, Dr. Francesca Patamello
and adhesion prevention
Vol. 69, No. 2, February
1998
for assistance in statistical analysis, and Dr. Valerie Colletta for linguistic assistance.
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