Table 1.
Study inclusion and exclusion criteria.
Figure 1.
On day 0, 2 sites on each upper arm were allocated (A). Sites were scanned using full-field laser perfusion imaging (FLPI) and spectrophotometric intracutaneous analysis (SIAscopy), before 5mm punch biopsies were taken under local anaesthetic (B). Excised tissue was processed using a range of laboratory techniques. Site 1 was left to heal by secondary intention, site 2 was filled with a 5mm disc of collagen-GAG scaffold (CG), site 3 treated with a 5mm disc of human decellularised dermis (DCD) while excised tissue from site 4 was replaced in the defect acting as an autograft (C). Subjects were seen weekly for 6 weeks where the wound was assessed clinically and non-invasive scanning repeated (D). Subjects subsequently underwent 6mm excision punch biopsies of healing tissue based on study group allocation (E). Excised tissue was again processed using a range of laboratory techniques. All wounds in the study healed satisfactorily leaving a small scar (F).
Figure 2.
74 volunteers were screened. 2 subjects did not fulfil recruitment criteria and were excluded. A further 22 declined to take part in the study. 50 participants were enrolled and randomly allocated to one of 5 groups each containing 10 patients. All groups had 4 punch biopsies harvested on day 0 before undergoing dermal skin substitute or control treatment. Group number determined the time point for excision biopsies of healing tissue. Group 1 at day 7, group 2 at day 14, group 3 at day 21, group 4 at day 28 and group 5 did not have repeat biopsies. Volunteers were reviewed weekly for 6 weeks post day 0 and wounds assessed serially using non-invasive measures. Complete data for each investigative modality at each time point was obtained with no loss to follow-up.
Table 2.
Antibody and staining regime used in CD31 immunohistochemical staining.
Figure 3.
Haemoglobin flux and oxyhaemoglobin concentration.
Change in haemoglobin flux (A) and oxyhaemoglobin concentration (B) after wounding in all treatment groups derived from full-field laser perfusion imaging (FLPI) and spectrophotometric intracutaneous analysis (SIAscopy) respectively. Non-invasive imaging confirms significantly up-regulated haemoglobin flux and oxyhaemoglobin concentration after treatment with DCD compared to controls. * p<0.05 n = 10/treatment group.
Figure 4.
Characteristic histological changes in acute cutaneous wounds left to heal by secondary intention (control) (2A-D) and treated with collagen-GAG scaffold (CG) (2E-H) focussing on blood vessel distribution and organisation. In both cases there is rapid development of granulation tissue with subsequent fibrosis, however CG samples develop fewer large vessels than controls. CG—collagen-GAG scaffold, E—epidermis, EB—epidermal bulge, F—subcutaneous fat, FD—fibrotic dermis, GT—granulation tissue, HS—hair shaft, ND—native dermis. Red arrow—patent vessel lumen, Black arrow—granulation tissue vessels. Black dotted line—demarcates border between normal tissue and healing tissue. Red dotted line—demarcates CG. Smaller image—x5 magnification, larger image—x20 magnification.
Figure 5.
Characteristic histological changes in acute cutaneous wounds treated using autografts (2A-D) and DCD (2E-H) focussing on blood vessel distribution and organisation. There is evidence of host migration through defined entry points in both grafted materials promoting revascularisation in autografts and rapid development of capillary networks in DCD. A—autograft, DCD—decellularised dermis, E—epidermis, EC—endothelial cells, Fb—fibroblast, FD—fibrotic dermis, GT—granulation tissue, ND—native dermis, RR—rete ridge. Red arrow—patent vessel lumen, Blue arrow—granulation tissue vessels, Orange arrow—dermal red blood cell extravasation. Black dotted line—border between autograft and host tissue, with finger like projections of inflammatory granulation tissue into autograft or DCD facilitating re-establishment of vascular channels. Smaller image—x5 magnification, larger image—x20 magnification.
Figure 6.
Changes in mRNA expression of prokineticin 2 (PROK2) (n = 5/treatment group) (A), membrane type 6 matrix metalloproteinase (MT6-MMP) (n = 5/treatment group) (B), hypoxia-induced translation factor 2A (HIF2A) (n = 5/treatment group) (C) and hypoxia-induced translation factor 3A (HIF3A) (n = 5/treatment group) (D) after cutaneous wounding derived from qRT-PCR. DCD promotes significant late up-regulation of PROK2 and MT6-MMP. * p<0.05 indicating significant differences between treatment groups at set time points.
Figure 7.
Immunohistochemical staining for endothelial cell marker CD31.
Alterations in CD31staining enabling calculation of vessel number after injury which was significantly greater in DCD after 28 days (C) (n = 10/treatment group). * p<0.05.
Figure 8.
Immunohistochemical evaluation.
Visualisation of time-matched (all d28) differences in CD31 expression between treatment groups in the same subject demonstrating significantly greater expression in the DCD treated wound. CD31 stain quantification assessed with Definiens Tissue Studio software and expressed as average vessel number/section. A—Control, B—CG, C—DCD, D—Autograft. Black dotted line—demarcates boundary between autograft and native tissue.