Histopathological Analysis of Decellularized Porcine Small Intestinal Submucosa after Treatment of Skin Ulcer

Abstract

Background: Decellularized porcine small intestinal submucosa (SIS), commercialized as an extracellular matrix rich in cell-inducing substrates and factors, has been clinically applied to treat intractable skin ulcers and has shown therapeutic effects. The SIS reportedly induces cell infiltration and integrates with the ulcer bed after 3-7 days of application. The attached SIS degenerates over time, and the remaining mass appears as slough, below which is granulation tissue that is essential for healing. This study aimed to determine whether the slough should be removed in clinical settings.

Methods: Five patients with intractable skin ulcers were included in this case series. Seven days after applying a two-layer fenestrated-type SIS to the ulcer, the removed slough was histopathologically examined.

Results: The collagen fibers of the SIS somewhat degenerated, and inflammatory cell infiltration was observed from the ulcer side to the surface side of the SIS. Neovascularization was similarly observed on the ulcer side. The degree of inflammatory cell infiltration decreased from the ulcer side to the surface side, whereas pus (ie, aggregates of neutrophils) was observed on the surface and ulcer edges. Additionally, the removed slough contained regenerative epithelium on the ulcer side of the remaining collagen fibers.

Conclusions: After treating intractable skin ulcers using SIS, we recommend removal of the upper surface and ulcer edge of the degenerated SIS or slough to prevent infection and preservation of the lower side of the degenerated SIS to maintain the granulation tissue and regenerative epithelium.

Fig. 1.

From SIS attachment to slough retrieval in Case 1. A, Skin ulcer before treatment. Good granulation can be observed on the ulcer bed. B, SIS attachment. SIS was applied to the ulcer, and nonadherent coating material was layered on the SIS. C, Status 1 week after SIS application. SIS degraded and red granulation was visible at the center. The area of SIS that retained its structure was not in contact with the ulcer bed. D, Harvested slough 7 days after SIS application. E, Ulcer after slough removal, showing decreased size due to epithelium extending from the ulcer edge.

Fig. 2.

Histopathologic analyses of slough and regenerative epithelium. A, Slough of Case 5. Inflammatory cells diffusely infiltrated into the slough and remained on its surface side. B, Slough of Case 5. The slough consisted of degenerative collagen fibers with diffuse inflammatory cell infiltration. Collagen fibers are stained blue by A/M stain. C, Slough of Case 3. A part of SIS-derived collagen fibers scarcely degraded, in which little cellular infiltration was observed. Inflammatory cells passed through SIS and accumulated on the surface. Collagen fibers are stained blue by A/M stain. D, Epithelialization of skin ulcer in Case 4. Slough was removed from ulcer 7 days after SIS application. Epithelium extension was observed from the ulcer edge. Complete structure of epithelium, from the basal layer to the stratum corneum, was observed histopathologically (inset: H/E). E, Slough of Case 4. Regenerative epithelium was observed on the ulcer side of mildly degraded SIS-derived collagen fibers. Squamous epithelium is stained red and collagen fibers are stained blue by A/M stain. The epithelium lacked a basal layer, had faintly-stained nuclei, and revealed parakeratosis (inset: H/E).

Fig. 3.

Immunohistochemical analyses of cells infiltrating in the slough. A, Mesenchymal cells, which express vimentin, are densely distributed on the ulcer side and migrated towards the surface side (Case 5). B, Mesenchymal cells diffusely infiltrated in the degraded collagen fibers (Case 1). C, Diffuse macrophage infiltration was observed in the slough. (Case 5). Macrophages express CD68. Cell density decreased from the ulcer side to the surface side. D, Pus in the slough of Case 1. Pus was observed on the ulcer edge and surface side of the slough. Neutrophils express CD33. E, Neovascularization on the ulcer side of the SIS (Case 1). Vascular endothelial cells express CD31. F, Neovascularization on the ulcer side of the SIS (Case 1). The microvessels contain smooth muscle cells, suggesting that they are either arteriole or venule. Smooth muscle cells are positive for αSMA.