Tissue reactivity and degradation patterns of absorbable vascular ligating clips implanted in peritoneum and rectus fascia Nasser Chegini,”$Stephen A. Metz,t and Byron J. Masterson* Department of Obstetrics and Gynecology, School of Medicine, University of Louisville, Louisville, Kentucky 40292 Absorbable vascular ligating clips are finding increasing use in intraabdominal surgery. We report the results of a light and scanning electron microscope investigation of the tissue reactivity and clip degradation patterns of two such materials, Absolok (polydioxanone) and Lactomer (copolymer of glycolic and lactic acid), implanted in the fascia and peritoneum of rabbits for intervals of 2 to 70 days. Cellular response to the clips, defined as the number of inflammatory cells/104 gm2, was maximum at day 4 postimplantation, then gradually declined as the duration of implantation increased. This pattern, seen

with both types of clips, was similar to that seen with polydioxanone (PDS) suture, but significantly greater than that associated with polypropylene (Prolene) suture. Although cellular response to the clips was greater in peritoneum than in fascia, especially on two occasions associated with adhesion formation, this was not statistically significant. Based on our morphological observations, the signs of clip degradation which were indicated by the appearance of surface crazing and cracks occurred earlier in peritoneum than in fascia.

INTRODUCTION

Mechanical staples are being used increasingly for a wide range of surgical procedures, including gynecologic surgery. Use of these devices for such applications as closure of the vaginal vault during abdominal hysterectomy has been associated with decreased tissue trauma, lower rate of infection, and reduced granuloma formation at the vaginal cuff, with consequent reduction in length of hospital The permanence of stainless steel presents obvious drawbacks for certain application^.'-^ This prompted a search for an absorbable staple material. Two such materials have been developed: polydioxanone (a polymer of the monomer para-dioxanone) from which Absolok vascular ligating clips2and *Presentaddress: Department of OB/GYN, University of Florida, College of Medicine, J-294, JHMHC, Gainesville, FL 32610.

‘Present address: United States Naval Hospital, Department of OBIGYN, Oakland, CA 94627-5000. *To whom correspondence should be addressed. Journal of Biomedical Materials Research, Vol. 24, 929-937 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0021-9304/90/070929-09$04.00

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PDS sutures3 are made, and a copolymer of lactic and glycolic acids, from are manufactured. which various absorbable suture^^*^ and Lactomer clips'~28s We have previously reported the effects of tissue rea~tion,~ bacterial infection,6and the nature of the local environment under in vitro and in vim cond i t i o n ~on ~ the degradation of these clips. This study was undertaken to examine the effect of two different in vivo conditions (fascial and intraperitoneal) on the tissue reaction to and resulting degradation of Absolok and Lactomer absorbable ligating clips. MATERIALS AND METHODS

All the materials for light and scanning electron microscopy were purchased from Polysciences, Inc. (Warington, PA) and Structural Probe, Inc. (West Chester, PA). Two types of absorbable ligating clips were used in this study: Absolok (Polydioxanone, Ethicon, Inc., Somerville, NJ); and Lactomer (glycolic/lactic acid copolymer, United States Surgical, Norwalk, CT). Experiments were performed using female New Zealand white rabbits weighing approximately 2.5 kg. The animals were anesthetized with intramuscular xylazine, 5 mg/kg, and ketamine, 37.5 mg/kg. Anesthesia was maintained with intravenous pentobarbital. A vertical midline incision was made in the lower abdomen, and the skin and subcutaneous tissue were dissected off the fascia laterally to a distance of approximately 5 cm. Depending on the experiment, either Absolok or Lactomer clips were clipped to the external surface of the fascia at the lateral margin of the field of dissection. A midline incision was then made in the fascia and peritoneum for the extent permitted by the skin incision. A paramedial incision was made in the intraperitoneal surface approximately 5 cm lateral to the midline. Clips of the same material used on the fascia were then clipped to the intraperitoneal surface and also used to close this latter incision. The midline fascial incision was closed with interrupted simple sutures of polypropylene (Prolene, Ethicon, Inc., Somerville, NJ), and the skin was closed with stainlesssteel skin staples. After intervals of 2, 4, 7, 14, 21, 35, and 70 days, two rabbits of each group were sacrificed using intravenous injection of lethal doses of T61 euthanasia solution. The midline incisions were opened and the clips, with attached tissue, were excised from the fascial and peritoneal sites. Samples for scanning electron microscopy were prepared by gently teasing the tissue from the clip in order to expose a small area of the clip surface. The specimens were then fixed as previously de~cribed.~ For light microscopy all the tissue attached to the clips during implantation were removed from the clips and fixed for 3 h at 4°Cin 2.5% glutaraldehyde buffered with 0.1M cacodylate buffer (PH 7.3), then post-fixed in osmium tetroxide, dehydrated, and embedded in JB4 embedding material.' Sections 2-3 pm thick were prepared and stained with a methylene blue-basic fuschin stain prepared according to the JEMembedding kit instructions.

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Quantitative analysis of cell density in the light microscope sections was performed using the Bioquant V Computer Image Analysis System. The tissue adjacent to the clip surface and clamped between the two braces of the clips was examined and all inflammatory cells present were included in the cell count. Total area on each slide examined was 50,000 pm.’ Statistical analysis of the results was performed using Student’s t test or one-way analysis of variance, as appropriate. RESULTS

Tissue inflammatory response was defined as the concentration of inflammatory cells in the tissue surrounding the clip surface. The inflammatory cell density in the fascia and peritoneal tissue surrounding the Absolok and Lactomer clips is shown in Figs. la and lb, respectively. Between postoperative days 2 and 4, there was a statistically significant ( p < 0.001) rise in inflammatory reaction in fascia from approximately 20 cells/104 pm2 to 50 cells/104 pm2 for both Lactomer and Absolok. In the peritoneum, the reaction was similar to that seen in fascia for both Lactomer and Absolok during the implantation period. However, the reaction at day 2 of postimplantation was as high as day 4 in the case of Absolok. This reaction was significantly higher ( p < 0.05) than that seen with Lactomer at the same postimplantation day. This higher tissue reaction may be explained by its association with adhesion formation. The inflammatory reaction to the clips

DAYS POSTIMPLANTATION

(4

DAYS POSTIMPLANTATION

(b)

Figure 1. Quantitative determination of inflammatory cells in tissue surrounding the Absolok (A) and Lactomer (B) clips during different days of implantation in peritoneum (m) and fascia (@).

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CHEGINI, METZ, AND MASTERSON

gradually decreased after 7 days postimplantation and did not change thereafter during the rest of the implantation period in this study, which was up to 70 days. The inflammatory reactions to both type of clips were similar during the implantation period examined in this study (Figs. l a & lb). A significant increase in inflammatory response to Absolok was observed at day 7 (44 f 3 cells/104 pm2) and day 21 (19 f 1.5 cells/104 pm2) postimplantation (see Fig. la). These inflammatory reactions to Absolok clips were associated with development of a local infection at the site of implantation. Data points for day 7 and 21 presented in Fig. la are representative of noninfected inflammatory reaction to Absolok from one animal for each time point. Light microscope photographs of peritoneal and fascia1 tissue attached to and surrounding the Absolok and Lactomer clips at 4 days postimplantation are shown in Fig. 2. There is a high concentration of acute inflammatory cells in these tissues. By 14 days postimplantation, the inflammatory cell density is considerably reduced. Figure 3 shows light microscope photographs of similar sections at day 35 postimplantation. At the scanning electron microscope level, both Absolok and Lactomer clips showed the presence of few cells attached to their surface at day 2 postimplantation. However, as the length of implantation was increased, the number of cells encapsulating the clips gradually increased. The clips were totally encapsulated by 14 days postimplantation. Collagen bundles of varying lengths and diameters were also noted in the encapsulating tissues (Fig. 4). Based on our morphological observations the sign of clips degradation, indicated by the appearance of surface crazing and cracks occurred on both Absolok and Lactomer. These morphological modifications were the early sign of clips degradation. However, we did not observe any fragmentation and also total breakdown of these clips during the 70-day implantation period. There were also no differences in the pattern of clip degradation regardless of location of implantation, although clips implanted in peritoneum developed surface crazing earlier than in fascia. DISCUSSION

The application of absorbable polymers for flexible monofilament and braided sutures and for surgical staples has gained increasing acceptance in gynecologic surgery. The absorbable ligating clips used in this study, Absolok, a polymer of para-dioxanone, and Lactomer, a copolymer of lactic and glycolic acids, are very similar in composition to PDS2j3and polyglactin 910 sutures, respectively.lr4 These sutures have been studied extensively in many animal ~ t u d i e s .Physical ~,~ properties such as tensile strength, degradation behavior under various in vim and in vitro environmental conditions, tissue reactivity, and the effects of bacterial infection of these sutures have been evaluated e~tensively.~,~,~

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Figure 2. Light microscope photographs of tissue adjacent to Absolok (A&B) and Lactomer (C&D) clips implanted in peritoneum (A&C) and fascia (B&D) for 4 days. Note the presence of many acute inflammatory cells (darkly stained). Original magnification x 130.

We have previously reported the effect of tissue reaction, bacterial infection and in vivo and in vitro conditions on the absorption characteristics and mechanical strength of Absolok and Lactomer

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CHEGINI, METZ, AND MASTERSON

Figure 3. Light microscope photographs of tissue adjacent to the Absolok (A&B) and Lactomer (C&D) clips implanted in peritoneum (A&C)and fascia (B&D) for 35 days. Note a considerable reduction in the number of inflammatory cells seen in Figure 2 which are replaced by fibroblasts and other elements of connective tissue. Original magnification x 130.

The present study was designed to extend our investigation of the tissue reaction and degradation patterns of clips implanted in rabbit fascia, and to compare these results with clips implanted in the peritoneal cavity. Tissue response in both environments is qualitatively similar. As the initial reac-

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Figure 4. Scanning electron micrographs of Lactomer (A&B) and Absolok (C) implanted in peritoneum (A) and fascia (B&C) for 4 days (A&B) and 35 days (C). A gradual accumulation of encapsulating tissue (arrows in A & B) containing fibroblasts (small arrow in B) and collagen bundles of different diameter (C) can be seen as the length of implantation increased. Original , X60, (C) ~3000. magnifications: (A) ~ 8 5 (B)

tion resolved, there was an influx of fibroblasts and concurrent deposition of fibrous connective tissue, which was complete by approximately 3 to 4 weeks postimplantation.

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The tissue reaction to absorbable ligating clips is similar to that previously observed in studies of PDS sutures, which are also fabricated from polydioxanone." However, as was noted for absorbable sutures, the tissue reaction elicited by absorbable ligating clips was significantly greater ( p < 0.001) than that stimulated by the nonabsorbable synthetic suture material polypropylene." In two instances the attachment site of the clips on the peritoneal surface led to an adhesion formation. This event was also associated with much higher inflammatory response as compared to sites without. This may have been caused by the extent of damage done to the tissue during clip application. Several factors are known to influence adhesion formation such as tissue ischemia, tissue trauma, foreign bodies, hemorrhage, and some other factors.l2 The clips recovered during the period of 70 days of implantation were inert both in fascia and peritoneum. Scanning electron microscopy indicated a gradual accumulation of tissue attachment to the surface of the clips and little morphological alteration of clip surface. The pattern of tissue attachment and degradation was similar to that reported previ~usly.~ The tissue attachment to the clip surface may greatly influence their degradation, as the clips were still identifiable at the site of implantation even after 25 weeks po~timplantation.~~~'" Steckel et al.' have reported similar findings using Lactomer staples when applied in vaginal lumen; however, only fragmentation of the staples was observed after 6 weeks, indicating their degradation. In conclusion, the results indicated that both clips induce a mild inflammatory reaction. Based on our previous observations," the clips retain their strength during the first few weeks after implantation. These properties make them ideal devices for different surgical application. This research was funded in part by the Marie Louise Woodson Foundation of Louisville, KY. The authors would like to thank Landis Payne for her help with the preparation of the manuscript.

References 1. R. R. Steckel, H. W. Jann, D. Kaplan, R. M. Jakowski, and A. Schwartz, "Experimental evaluation of absorbable copolymer staples for hysterectomy," Obstet. Gynecol., 68, 404-410 (1986). 2. C. J. Schaefer, P. M. Colombani, and G. W Geelhoed, "Absorbable ligating clips," Surg. Gynecol. Obsfef., 154, 513-516 (1982). 3. J. A. Ray, N. Doddi, D. Regula, J. A. Williams and A. Melveger, "Polydioxanone (PDS), a novel monofilament synthetic absorbable suture," Surg. Gynecol. Obstet., 153, 497-507 (1981). 4. N. A. Swanson and T. A. Tromovitch, "Suture materials, 1980's: properties, uses and abuses," l n f . J. Derrnatol., 21, 373-378 (1982). 5. N. Chegini, J. A. von Fraunhofer, D. L. Hay, and B. J. Masterson, "Tissue reactions to absorbable ligating clips," 1, Xeprod. Med., 33, 187-192 (1988). 6. N. Chegini, D. L. Hay, J. A. von Fraunhofer, and B. J. Masterson, "The effects of bacterial infection on absorbable vascular ligating clips," J. Reprod. Med., 33, 25-29 (1988).

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N. Chegini, D.L. Hay, J.A. von Fraunhofer, and B. J. Masterson, "A comparative scanning electron microscope study on degradation of absorbable ligating clips in vivo and in vitro," 1. Biomed. Mater. Res., 22, 71-79 (1988). N. Chegni and Ch.V. Rao, "Quantitative light microscope autoradiographic study on L3H] Leukotriene C4 binding to nonpregnant bovine uterine tissue," Endocrinology, 122, 1732-1736 (1988). N. Chegni, "Absorbable ligating clips: Their ultrastructural characteristics in vivo and in vitro," in Clin. Obstet. Gynecol., Symposium on Gynecologic Surgery, B. J. Masterson (ed.), Lippincott, Philadelphia, -1988, V V . 725-735. D.L. Hay, J. A. von Fraunhofer, N. Chegini, and 8. J. Masterson, "Locking mechanism strength of absorbable ligating devices," 1. Biomed. Muter. Res., 22, 179-190 (1988). S . A. Metz, N. Chegioi, and B. J. Masterson, "In vivo tissue reactivity and degradation of suture materials. A comparison of Maxon and PDS," 1. Gynecol. Surg., 5(1), 37-46 (1989). J. H. Bellina, R. Hemmings, J. I. Voros, and L. E. Ross, "Carbon dioxide laser and electrosurgical wound study with an animal model: A comparison of tissue damage and healing patterns in peritoneal tissue," Am. J. Obstet. Gynecol., 148, 327 (1984). I S

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Received March 15, 1989 Accepted November 22, 1989

Tissue reactivity and degradation patterns of absorbable vascular ligating clips implanted in peritoneum and rectus fascia.

Absorbable vascular ligating clips are finding increasing use in intraabdominal surgery. We report the results of a light and scanning electron micros...
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