Histological analysis of fat grafting with platelet-rich plasma for diabetic foot ulcers-A randomised controlled trial

Abstract

Diabetic foot ulcers are often unresponsive to conventional therapy and are a leading cause of amputation. Animal studies have shown stem cells and growth factors can accelerate wound healing. Adipose-derived stem cells are found in fat grafts and mixing them with platelet-rich plasma (PRP) may improve graft survival. This study aimed to establish the histological changes when diabetic foot ulcers are treated with fat grafts and PRP. A three-armed RCT was undertaken of 18 diabetic foot ulcer patients: fat grafting; fat grafting with PRP; and routine podiatry care. Biopsies were obtained at week 0, 1, and 4, and underwent quantitative histology/immunohistochemistry (H&E, CD31, and Ki67). Treatment with fat and PRP increased mean microvessel density at 1 week to 1645 (SD 96) microvessels/mm2 (+32%-45% to other arms, P = .035). PRP appeared to increase vascularity surrounding fat grafts, and histology suggested PRP may enhance fat graft survival. There was no clinical difference between arms. This study demonstrates PRP with fat grafts increased neovascularisation and graft survival in diabetic foot ulcers. The histology was not, however, correlated with wound healing time. Future studies should consider using apoptosis markers and fluorescent labelling to ascertain if enhanced fat graft survival is due to proliferation or reduced apoptosis. Trial registration NCT03085550.

Keywords: adipose-derived stem cells; diabetic foot ulcers; fat grafting; histology; platelet-rich plasma.

Conflict of interest statement

The authors declare no conflicts of interest.

© 2021 The Authors. International Wound Journal published by Medicalhelplines.com Inc (3M) and John Wiley & Sons Ltd.

Figures

FIGURE 1

Methods used for quantification of microvessels in dermal tissue from skin biopsies, based on a standard technique for dermal tissue. A, shows three areas are identified visually as having the highest levels of CD31 uptake and therefore highest density of stained microvessels. Three non‐overlapping fields at x500 magnifications (red boxes) are selected. B, shows the left‐most area at higher magnification. The avascular epidermis has not been included in the area. Any brown staining was counted as a microvessel and 10 example microvessels have been marked with a red flag. A lumen was not required to be identified as a microvessel, three example lumens of vessels have been marked with red circular markers for alliteration purposes. Vessels with a thick muscular wall (none shown) or a diameter > 50 μm (marked by black arrow) were excluded from the count. The microvessel count from this particular area was 44, 36, and 49. As this has a > 10% variation it was counted a further two times (41, 45) and a mean taken of all readings (mean = 43). The area was calculated automatically by the software (NPD.view) at 0.0286 mm2. This gave a microvessel density of 1503 microvessels/mm2

FIGURE 2

A to F, Histology from an example patient who underwent treatment with fat grafting mixed with PRP. The above samples were all taken at 1‐week post‐intervention from the edge of a diabetic foot ulcer. A, is stained with haematoxylin and eosin (H&E) and mature adipocytes can be seen in the bottom left of the image next to the black arrow. B, is the same section, from the same patient at the same time point stained with CD31 (an endothelial marker for blood vessels). The blood vessels in the reticular dermis stain brown/black and can be visualised in line with the red arrow. The epidermis is avascular. There also appears to be increased CD31 uptake around the adipocytes in the bottom left where the sample has been treated with fat grafting and PRP. C, is the lower left box from B in higher magnification, and D, is the box in C magnified. Individual microvessels can be seen, which have formed around the adipocytes. There is also increased uptake of CD31 generally amongst the adipocytes suggesting there are many more microvessels that are too small to be fully appreciated. Several microvessels as examples have been labelled with red arrows; however, similar small brown lumens can be seen around every adipocyte. E, in contrast is the lower right box from B and shows an area where there was not grafted fat/PRP. There is a paucity of microvessels in this field of view. Instead there are two large mature vessels that can be seen transected in the box (marked with red arrows). There is less uptake of CD31 throughout also, again suggesting a lack of microvessels. F, is stained with Ki67 (cellular proliferation. Uptake of Ki67 can be seen primarily at the dermal‐epidermal junction and is occurring in the basal layer of the epidermis. Otherwise there is a similar proportion of Ki67 positive staining cells throughout the sample and there is no association with the grafted fat/PRP in the bottom left of the slide. G and H are H&E staining of a central wound biopsy in a patient in the fat grafting mixed with PRP study arm, 1 week following treatment. The adipocytes from the fat graft are shown near the back arrow. A large number of adipocytes have survived and they appear well organised. H, is the area by the back arrow at higher magnification where the organisation be more clearly appreciated. I to K, are H&E staining of a central wound biopsy in a patient in the fat grafting only study arm, 1 week following treatment. Mature rounded adipocytes are again visible at the peripheries of the central biopsy (black and blue arrows). In comparison to the fat/PRP sample above, the adipocytes are less densely packed, less numerous, and less well organised. This suggests that survival has not been achieved by as many grafted cells. J and K are higher magnification by the black and blue arrows, respectively. At higher magnification the lower density of adipocytes and lower level of organisation can be more easily appreciated. More pink staining tissue can be seen between cells in this slide compared with the fat/PRP sample

FIGURE 3

A boxplot of epithelial thickness (mm) from H&E staining at three time points in the different study arms following the various interventions. There was no statistically significant difference between any of the groups at any time point (P = .115). Not all samples could be included at every time point due to some being oblique sections and therefore unsuitable for this analysis

FIGURE 4

A boxplot of microvessel density (vessels per mm2) at time points in the different study arms following the various interventions. A statistically significant difference was seen at 1‐week post‐intervention between the three treatment groups (P = .035). No significant difference in microvessel density was observed between the groups at the time of intervention (week 0, P = .797) and 4 weeks post‐intervention (P = .152). When analysed by intervention across the three time points, fat/PRP trended towards statistical significance (P = .071). The remaining P values for fat only were .103 and for controls were .926

FIGURE 5

The proportion of cells staining positive for Ki67 by week and intervention. There was a statistically significant change in the control group over time (P = .047). There was no difference in either of the intervention groups across the different time points (P = .338 for fat only, P = .304 for fat/PRP). When analysed by week there was also no difference (week 0 P = .221, week 1 P = .231, week 4 P = .05)