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. 2013 Dec 18:4:2041731413518060.
doi: 10.1177/2041731413518060. eCollection 2013.

Porcine cholecyst-derived scaffold promotes full-thickness wound healing in rabbit

Deepa Revi  1 , Vadavanath Prabhakaran Vineetha  1 , Jaseer Muhamed  1 , Akhila Rajan  1 , Thapasimuthu Vijayamma Anilkumar  1
Affiliations

Porcine cholecyst-derived scaffold promotes full-thickness wound healing in rabbit

Deepa Revi et al. J Tissue Eng. .

Abstract

Graft-assisted healing is an important strategy for treating full-thickness skin wounds. This study evaluated the properties of porcine cholecyst-derived scaffold and its use for treating full-thickness skin wound in rabbit. The physical properties of cholecyst-derived scaffold were congenial for skin-graft application. Compared to a commercially available skin-graft substitute made of porcine small intestinal submucosa, the cholecyst-derived scaffold was rich in natural biomolecules like elastin and glycosaminoglycans. When used as a xenograft, it promoted healing with excess cell proliferation at early phases and acceptable collagen deposition in the later remodelling phases.

Keywords: Wound healing; cholecyst-derived scaffold; collagen; extracellular matrix; graft-assisted healing; skin graft.

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Conflict of interest statement

Declaration of conflicting interests: The authors declared no potential conflicts of interest with respect to the research, authorship and publication of this article.

Figures

Figure 1.
Figure 1.
Physical properties of the scaffold: (a) moisture content, (b) fluid uptake, (c) evaporative water loss (EWL), (d) water vapour transmission rate (WVTR), (e) flexural rigidity and (f) suture retention strength. The CDSs have lower WVTR (p value = 0.001) and suture retention strength (p value = 0.003) than SIS. CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa.
Figure 2.
Figure 2.
(a) Content of biomolecules in the candidate skin graft: the CDSs have higher elastin (p value = 0.0009) and sulphated GAG (p value = 0.006) than SIS. (b) DNA content in the scaffold: CDSs have lower DNA (p value = 0.03) compared to SIS. CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa.
Figure 3.
Figure 3.
Haematoxylin and eosin staining of (a, c, e and g) CDS-grafted wound and (b, d, f and h) SIS-grafted wound on 3rd, 7th, 14th and 30th day. CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa.
Figure 4.
Figure 4.
PCNA immuno staining of CDS-grafted wound (a,c,e and g) and SIS-grafted wound (b,d, f and h) on 3rd, 7th, 14th and 30th day. On third day (a and b), most of the basal cells of neo-epidermis (black arrow) and the dermis immediately beneath are positive for PCNA. During 7th day (c and d), the outer layers of epidermis remained negative, but the basal and suprabasal cells in the neo-epidermis show PCNA-positive in both grafts. By 14th (e and f) and 30th day (g and h), only basal cells (white arrow) in epidermis were positive for PCNA. Please see Figure 11(c) for quantitative data. CDS: cholecyst-derived scaffold; PCNA: proliferating cell nuclear antigen.
Figure 5.
Figure 5.
CK-14 immuno staining of CDS-grafted wound (a,c,e and g) and SIS-grafted wound (b,d, f and h) on 3rd, 7th, 14th and 30th day. On 3rd (a and b) and 7th day (c and d), CK-14 deposition can be seen on the leading edge of re-epithelisation (arrow) and the suprabasal keratinocytes of the epidermis (white star) in both CDS- and SIS-grafted wounds. On day 14 (e and f) and day 30 (g and h), the epidermis was completely formed and CK-14 deposition was seen in the middle layers of epidermis (black star) in wounds treated by both the grafts. CK-14: cytokeratin-14; CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa.
Figure 6.
Figure 6.
Vimentin immuno staining of CDS-grafted wound (a,c,e and g) and SIS-grafted wound (b,d, f and h) on 3rd, 7th, 14th and 30th day. On third day (a and b), both CDS and SIS showed heterogeneous distribution of vimentin throughout the wound area and they appeared like elongated spindles (star). On 7th (c and d) and 14th day (e and f), the vimentin concentrated below the neo-epidermis showed further elongation and scattering. On 30th day (g and h), vimentin deposition was sparse, faint, thin and assumed the shape of an ‘elongated wavy thread’ (arrow). Please see Figure 11(d) for quantitative data. CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa.
Figure 7.
Figure 7.
ASMA immuno staining of CDS-grafted wound (a,c,e and g) and SIS-grafted wound (b,d, f and h) on 3rd, 7th, 14th and 30th day. On third day (a and b), ASMA was distributed throughout wound area and showed spindle, elongated and wavy pattern (arrow). On 7th day (c and d) and 14th day (e and f) ASMA further increased (star) and was concentrated below the newly formed epidermis and at the wound–tissue interface in the dermis. During day 30 (g and h), ASMA concentration decreased compared to day 14. Please see Figure 11(e) for quantitative data. CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa; ASMA: anti-smooth muscle antibody.
Figure 8.
Figure 8.
Picro-sirius red staining of (a, c, e and g) CDS-grafted wound and (b, d, f and h) SIS-grafted wound on 3rd, 7th, 14th and 30th day. Collagen deposition increased from 3 to 7 days. On 14th (e and f) and 30th day (g and h), the collagen fibres were elongated and closely arranged (star) than 3rd (a and b) and 7th day (c and d) when collagen fibres are seen as bundles (arrow). Please see Figure 11(f) for quantitative data. CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa.
Figure 9.
Figure 9.
Herovici staining of (a and b) CDS-grafted wound and (c and d) SIS-grafted wound on 7th and 30th day. The CDS-grafted wound showed higher type I collagen deposition (red fibres) on 30 days (b and d) compared to SIS-grafted wounds. Please see Figure 12(a) for quantitative data. CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa.
Figure 10.
Figure 10.
Van Gieson staining of (a and b) CDS-grafted wound and (c and d) SIS-grafted wound on 7th (a and c) and 30th day (b and d). Note that the elastin deposition (black fibres) increased on 30th day (b and d) in both CDS- and SIS-grafted wounds. Please see Figure 12(b) for quantitative data. CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa.
Figure 11.
Figure 11.
Quantitative histomorphometry for early stage wound-healing parameters: (a) percentage re-epithelisation, (b) angiogenesis, (c) cell proliferation in dermis, (d) area occupied by vimentin-positive cells, (e) area occupied by alpha smooth muscle actin positive cells and (f) collagen deposition. The CDS-grafted wound showed higher ASMA-positive area on 3rd day (p value = 0.009) as well as cell proliferation on 3rd (p value = 0.02) and 14th day (p value = 0.008). CDS: cholecyst-derived scaffold; ASMA: anti-smooth muscle antibody.
Figure 12.
Figure 12.
Quantitative data for late-stage wound-healing parameters: (a) ratio of collagen type I to collagen type III ratio, (b) elastin deposition. CDS had higher type 1 to type III collagen ratio compared to SIS on 30th day (p value = 0.005). CDS: cholecyst-derived scaffold; SIS: small intestinal submucosa.
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