732 Collagen content No significant differences were observed in the anastomotic regions in terms of relative collagen areas (collagen content expressed relative to total tissue area) and red versus green ratios (collagen maturity) between groups at 3 and 7 days follow-up (Table 1). All groups combined, relative collagen areas at the anastomotic region (6.1 ± 1.6%) were signifi- cantly lower than at the perianastomotic region at 3 days follow-up (15.4 ± 2.3%, P= 0.001), but equal at 7 days follow-up (18.9 ± 3.3% vs. 18.3 ± 3.0%, P= 0.826). This was due to a significant increase in the relative collagen area at the anastomotic region between 3 (6.1 ± 1.6%) and 7 days follow-up (18.9 ± 3.3%, P= 0.001). Histology Anastomotic regions in the control and fibrin glue groups showed a similar acute inflammatory response at 3 days follow-up, characterized by necrosis and granulocyte infiltration (Fig. 3a). Fibrin glue was still microscopically detectable at 3 days follow-up. However, application of a collagen fleece induced a more severe inflammatory reaction with deep necrosis at the fleece–bowel interface (Fig. 3c). At 7 days follow-up, the control and the fibrin glue group reached a chronic inflammatory state with the formation of granulation tissue and the infiltration of macrophages and lymphocytes (Fig. 3d, e). In addition, the first signs of epithelial cells migrating from the margins of the wound to the defect became apparent (Fig. 3e). However, anastomoses with a collagen fleece still showed a more severe inflammation than the other groups with necrosis and a high density of inflammatory cells, including granulocytes, macrophages, and lymphocytes (Fig. 3f). Discussion In the present study, no beneficial effect of a collagen fleece on anastomotic healing was observed compared to control Int J Colorectal Dis (2011) 26:729–735 groups. Bursting pressures and collagen content were not improved, but instead, bowel obstructions and increased inflammation occurred significantly more often in the collagen fleece group. The return in strength after a gastrointestinal anastomosis is considered to be mainly dependent on the formation of collagen [16]. However, early after the construction of an anastomosis, degradation of the extracellular matrix occurs [17, 18]. This was observed at 3 days follow-up when a lower collagen content in comparison with the perianastomotic region was found. Thereafter, the remodeling phase begins and the collagen content increases, as observed at day 7. Together with the crosslinking of thin, immature collagen fibers, these processes are generally regarded to improve the anastomotic strength. The strength of the anastomosis can be reliably quanti- fied by examining the bursting pressure [19]. Also, the thicker fibers can be distinguished from the thinner fibers by cross polarization microscopy after picro-sirius red staining. In this study, however, both the bursting pressures as well as the collagen maturation were not improved following fibrin glue and collagen fleece application to the anastomoses. Besides the absence of any improvements in anastomotic strength, bowel obstructions occurred significantly more often in the collagen fleece group. Although unexpected, other experimental studies have confirmed the occurrence of obstructions after wrapping a fleece of collagen or dura mater around a bowel anastomosis [20, 21]. The exact mechanism remains unclear, but shrinkage of the collagen fleece can be severe and could mechanically impair normal passage of stool, especially in the colon [22]. Additionally, the external constriction by the fleece together with the observed increased inflammatory reaction might induce a more pronounced inward bulging of the edematous colonic wall. A proposed mechanism with increased fibrosis is unlikely since collagen contents did not differ significantly between groups in this study [20]. Yet, it must be noted that this study only displays the results at 3 and 7 days follow-up. After full resolution of the collagen fleece, the colonic wall Table 1 After picro-sirius red staining of the anastomotic region, the relative collagen area was quantified as the percentage of total tissue surface occupied by collagen 3 days follow-up 7 days follow-up Relative collagen area Red/green ratio Relative collagen area Red/green ratio Control 8.5±2.7% 1,195.4±834.0 25.7±7.8% 963.0±468.5 Fibrin glue 4.4±3.7% 398.1±279.2 17.3±4.0% 1,904.5±707.4 Collagen fleece 4.9±2.0% 227.2±75.3 14.6±3.3% 1,664.5±547.2 P value 0.518 0.456 0.336 0.507 Maturity of collagen was estimated by calculating the red versus green ratio where red indicates thick, more mature collagen fibers and green indicates the thinner, immature collagen fibers. No significant differences were observed between groups at either 3 or 7 days follow-up All measurements in this table are mean (SEM)