MATERIALS AND METHODS

Experiment design: Twenty seven male Mongrel dogs (30-35 kg b.wt.) were randomly allotted to constitute 3 groups (1, 2 and 3) having 9 animals each. All dogs were premedicated with atropine sulphate (0.04 mg kg^-1, I.M) and xylazine hydrochloride (2 mg kg^-1, I.M). General anesthesia is achieved by 5% thiopental sodium (10 mg kg^-1, I.V.). Typhlectomy was performed as per standard aseptic procedure as described by Slatter (2003). The colonic wounds in dogs from group 1, 2 and 3 were sutured with 3/0 chromic catgut, 3/0 non-absorbable braided silk and 3/0 polyglactic acid 910 (polyglactin/Vicryl), respectively. Omentalization of the colonic wound was performed as per standard procedures. Post-operative dressing of surgical wounds along with antibiotic and anti-inflammatory coverage was given for 3 Post-Operative Days (POD). On the 7th, 14th and 21st POD, three dogs from each group were euthanized by intravenous injection of an overdose of thiopental sodium 5% for studying the gross and histopathological parameters. All the animals were handled, maintained and disposed according to the experimental animal ethics approved by South Valley University, Qena, Egypt.

Clinical examination: Rectal temperature, respiratory and heart rates of each dog were recorded before surgery and also for 3 successive POD. Abdominal skin sutures were also observed daily for wound dehiscence, edema or swelling and suppuration for 7 successive POD.

Gross examination of the colonic wound: The colonic wound adhesions with omentum and other tissues such as mesenteric lymph nodes and other parts of the intestine were grossly assessed and graded using a standard scale from 1-3; one for mild adhesion at one point of the wound with a fibrous band. Two for a moderate adhesion occurred at more than one point and 3 for severe adhesion almost circumscribed entire colonic closure. Moreover, the colonic wound stump was investigated for dehiscence, leakage and presence of suture material. The height and length of the mucosal ridges, formed at the site of the colonic wound closure and the time of their disappearance as well as the presence of suture material were also recorded.

Microscopical examination of the colonic wound: Tissue specimens were obtained from the junction of the colonic wound and immersed in 10% neutral formalin for fixation. The tissue specimens were processed as per standard histopathological procedures and embedded in paraffin wax. Sections of 5 μ thickness were prepared and stained by haematoxylin and eosin. Organization of wounds was studied by staining tissue sections with Van Gieson stain.

Table 1:	Description and scoring of histopathological changes of the colonic wound healing
*Degree of apposition of the colonic edge, **Degree of integrity of the epithelium overlying the colonic wound

The following histopathological changes were studied, scored and recorded in all animals (Table 1):

Statistical analysis: The severity of adhesions between the colonic wound and omentum and the histopathological examination was statistically analyzed between groups by using the survival (event) analysis test. Mean height and length of ridges were subjected to test of significance by one-way ANOVA wherein, mean values of height and length of ridges among all groups were compared by Dunnett’s test using Graphed Pad Prism (Graphed Pad software, San Diego, CA, USA). Results were considered significant where p<0.05.

RESULTS

All animals after typhlectomy were survived and had no abnormal signs from the 2nd POD till the end of the experiment. The mean of the temperature, respiratory and heart rates were 38.6±0.55°C, 39±8 and 106±16 min^-1, respectively.

Gross findings: The degree of adhesion of the colonic wound to the omentum varied among the 3 groups. Almost circumscribed severe adhesions were observed in animals from group 1. Moderate adhesions, more than one point, were observed in animals of group 2 whereas, mild adhesions, almost one point, were observed in animals from group 3. The statistical analysis revealed highest degree of surgical wound adhesion with omentum in animals from group 1 followed by those of group 2 and 3 in a decreasing order, however, the differences were statistically non-significant (Fig. 1). Similarly, non-significant differences in adhesions with the surrounding tissues were observed among the groups wherein, highest prevalence of adhesions was in animals of group 1 and 2 as compared with those of group 3 (Table 2). Wound dehiscence was not observed in all groups except one dog in group 1 at the 7th POD. Presence of suture material was recorded almost on the serosal surface in the 3 groups.

Fig. 1:	Histogram illustrating adhesion of the colonic stump to the omentum and other tissues

Table 2:	Prevalence of adhesion of colonic wound to intestine and other tissues using 3 types of suture material at the 7th, 14th and 21st POD

Table 3:	Mean height and length (mm) of the mucosal ridge (±SD) after colonic wound closure using 3 different suture materials

The mean and standard deviation values of heights and lengths of the colonic mucosal ridges have been depicted in Table 3. Mean heights of the ridges among all groups and also at different POD did not differ significantly. The mean length of mucosal ridges in animals of group 2 also did not differ significantly throughout the experiment but in animals of groups 1 and 3, the length of the ridges at the 21st POD was significantly lower than those at the 7th and 14th POD. The remnant of the suture materials on the mucosal surface of the colonic wound were seen in group 2 but not in group 1 and 3.

Microscopic findings: At the 7th POD, the mucosal fold was severely necrosed with less epithelisation in group 1 and 2 (Fig. 2a). Moreover, the tunica muscularis was moderately to severely destroyed, where extensive areas of severe necrosis and inflammatory cell infiltration between muscle fibers were observed. The suture material was surrounded by large granulomas composed of numerous cellular infiltrations with fibroblastic capsule and the surrounding tissue was severely destroyed. However, a mild to moderate mucosal necrosis with mild small cellular granulomas composed of moderate inflammatory cells, in addition to mild destruction to the tunica muscularis, were seen in group 3.

At the 14th POD, a moderate degree of mucosal necrosis with active process of epithelisation (Fig. 2b) and multifocal areas of inflamed tunica muscularis were observed in groups 1 and 2. Moderate to severe tissue reaction around the suture materials was also observed (Fig. 2d, e). In contrast, animals of group 3 revealed minimal mucosal necrosis with complete epithelialisation of the mucosa on the colonic wound (Fig. 2b, c). Thin layer of mild fibroblastic proliferation with minimal cellular infiltration was observed around the suture material (Fig. 2f). The destruction incurred to tunica muscularis was mild.

At the 21st POD, intact mucosal layer with good epithelisation and minimal cellular infiltration in the lamina properia was observed. Remnant of suture material surrounded by large cellular granulomatous and fibrous connective tissue with destruction of the tunica muscularis was observed in groups 1 and 2 (Fig. 2g, h). The destruction of tunica muscularis was subsided at this stage. In group 3, apposition with complete epithelialisation and minimal cellular infiltration at the wound edges of the mucosal layer was observed. The destruction of tunica muscularis and the tissue reaction were almost minimal at this stage (Fig. 2i).

Fig. 2(a-i):

(a) Mucosal necrosis and incomplete apposition of wound edges with severe inflammatory cellular infiltration in submucosa were observed at 7th POD in group 1 (H and E, 4X, 10X, 20X), (b) Severe necrosis in the mucosal fold of the free edge projection in the intestine lumen at 14th POD in group 2 compared with almost intact mucosa in the continued wound edge (H and E, 4X, 10X, 20X), (c) Almost intact mucosal fold with minimal inflammatory cell infiltration in lamina properia at 14th POD in group 3 (H and E, 4X, 10X, 20X), (d) Severe inflammatory cell infiltration mainly lymphocytes and macrophages with proliferation of fibroblast was observed around the remnant of the suture material at 14th POD in group 1 (H and E, 4X, 10X, 20X), (e) Tunica muscularis were severely destructed with remnant of suture materials. The latter surrounded by lymphocyte, macrophages and fibroblast proliferation at 14th POD in group 2 (H and E, 4X, 10X, 20X), (f) Threads of suture material in serosal layer with minimal cellular reaction at the 14th POD in group 3 (H and E, 4X, 10X, 20X), (g) Severe destruction of tunica muscularis replaced by fibrous connective tissue and more cellular reaction at 21st POD in group 1 (Van Giesson, X4), (h) Remnant of suture material is surrounded by cellular granuloma and fibrous connective tissue at 21st POD in group 2. Note destruction of the tunica muscularis (Van Giesson, 4X, 10X, 20X) and (i) Remnant of suture material is surrounded by minimal cellular reaction. Note the condensation of the submucosa at 21st POD in group 3 (Van Giesson, 4X, 10X, 20X)

The scores of mucosal necrosis, wound discontinuity, granulation tissue formation and destruction of tunica muscularis were non-significantly different among the 3 groups whereas, tissue reaction around suture materials was significantly (p<0.05) higher in group 1 as compared to that in group 2 and 3 in a decreasing order.

Fig. 3(a-e):	Statistical analysis of the scores of the histopathological picture of the colonic wound healing after using three types of suture materials. *Significant difference (p<0.05)

Minimal necrosis, destruction of tunica muscularis and granulation tissue formation with good epithelialisation were observed in group 3 (Fig. 3).

DISCUSSION

Surgery of the large intestine is associated with many complications such as wound dehiscence, leakage, adhesions and peritonitis (Borenstein et al., 2001). The cecum, as a part of the large intestine, may also have the same complications during its surgery. In the present study, silk was selected as a good example for a natural non absorbable suture material. Whereas, chromic catgut and vicryl as natural and synthetic absorbable suture materials, respectively were investigated to evaluate the most suitable/preferred suture material for closing colonic wound during typhlectomy. Therefore, the operation was performed by one surgeon and one assistant and both of them were comprehensively trained on typhlectomy before starting the experiment.

Hedlund (2002) and Brown (2003) recommended omentalisation immediately after performing intestinal surgery for prevention of leakage of intestinal chime. Therefore, the adhesions of the colonic wound to the omentum observed in the present study are preferred outcomes rather than a post-operative complication because omentalisation was applied during the surgical operation.

Wound dehiscence might play a second role in adhesions formation (Crowe and Bjorling, 1993), but in the present study, only one dog exhibited post-surgical colonic wound dehiscence. Therefore, adhesions observed in other dogs might be attributed to roughening of the serosal surface caused by presence of suture materials thereon. In the present study, severe adhesions were observed in dogs where chromic catgut and silk were used for closing colonic wound in groups 1 and 2, however, mild to moderate adhesions were observed in group 3 when using vicryl for wound closure. These findings are in agreement with those reported by Bennett (1988) and Akinrinmade and Lawal (2010).

In the present study, remnants of silk were found on the mucosa of the colon at the 21st POD that may result in permanent intra-luminal ridge formation, but this hypothesis requires further extensive study for confirmation. Moreover, presence of silk remnant on the mucosal surface of the colonic wound indicates that the healing process go towards extrusion of suture material rather than capsulation that in turn may lead to ulceration around the intra-luminal hanged suture material which may act as a nidus for fecal matter deposition and subsequent faecolith formation which could lead to intestinal obstruction (Pecha et al., 1998).

Dogs in group 3 showed a minimal necrosis in the mucosa, complete apposition of the wound edges with complete epithelization of the mucosal layer, regenerated submucosa and minimal cellular infiltrations surrounding the remnant of suture materials at the serosal layer. However, dogs in groups 1 and 2 have moderate to severe necrosis of the mucosal folds with partial epithelization, slightly thickened submucosa with granulation tissue related to the wound edges and a severe inflammatory cell reaction surrounding remnant threads of the suture materials in serosa. The healing process and the inflammatory reacation observed around the remanats of suture materials corroborates with the findings reported by Bennett (1988) and Bellenger (1982).

Chromic catgut is digested or phagocytosed by cellular elements in the blood that leads to engorgement of blood vessels at the site of healing with subsequent more tissue reaction (Greenwald et al., 1994). Silk threads act as a foreign material thereby incites encapsulation (Grabb, 1991) whereas Vicryl has been reported to get hydrolyzed into natural body tissues metabolites (Yaltirik et al., 2003).

On the basis of clinical, surgical and histopathological evaluation of silk, catgut and Vicryl for closing the colon wound during typhlectomy, Vicryl was found to be the most suitable suture material because it bears sufficient tensile strength required for closure of colonic wound. In addition, it causes least necrosis and better epithelization, mild to moderate adhesions with surrounding tissues and minimal cellular infiltration/tissue reaction as compared with silk and catgut.

ACKNOWELDGMENT

We would like to express our deep thanks to Dr. Sadan, M.A. lecturer of Surgery for his technical help and Dr. Ahmed Ezzat, lecturer of Theriogenology for statistical analysis of this study.