Microvascular Fragment Transplantation Improves Rat Dorsal Skin Flap Survival

Abstract

Background: The development of flap necrosis distally remains a concern during microsurgical flap transfers because, at least in part, of decreased perfusion. Microvascular fragments (MVFs) are microvessels isolated from adipose tissue that are capable of improving tissue perfusion in a variety of tissue defects. The aim of this study was to determine whether the transplantation of MVFs in a dorsal rat skin flap model can improve flap survival.

Methods: A 10 × 3 cm flap was raised in a cranial to caudal fashion on the dorsal side of 16 Lewis rats, with the caudal side remaining intact. The rats were equally divided into a treatment group (MVFs) and a control group (sterile saline). At the time of surgery, sterile saline with or without MVFs was injected directly into the flap. Microvessel density was determined after harvesting flap tissue by counting vessels that positively stained for Griffonia simplicifolia lectin I-isolectin B₄. Laser Doppler was used to measure blood flow before and after surgery and 7 and 14 days later. Flap survival was evaluated 7 and 14 days after surgery by evaluating the percentage of viable tissue of the flap with photodigital planimetry.

Results: Despite the lack of a significant difference in microvessel density and tissue perfusion, flap survival increased 6.4% (P < 0.05) in MVF-treated animals compared with controls.

Conclusions: The use of MVFs may be a means to improve flap survival. Future studies are required to delineate mechanisms whereby this occurs and to further optimize their application.

Fig. 1.

Schematic depicting the MVF isolation procedure and injection into the dorsal skin flap. A and B, Adipose tissue was harvested, minced, digested with collagenase, centrifuged (400g × 4 min), and (C) filtered through 500 μm and 30 μm filters to (D) isolate a heterogeneous mixture of MVFs. Black scale bar represents 100 μm. E, View of dorsal side of the rat with a diagram of the 10 × 3 cm skin flap. Dashed lines indicate edges that were cut and flap was raised (blue arrow) in a cranial to caudal fashion with the caudal side remaining intact. Injections were performed by injecting MVFs in 10 evenly dispersed sites in the distal half. Control animals underwent the identical surgery with sterile PBS injected alone as a control. F, Diagram of the 3 regions (distal, middle, and proximal) into which the flap was separated for perfusion and microvessel density measurements.

Fig. 2.

A, Digital images of PBS- and MVF-treated dorsal skin flaps 7 and 14 days after surgery. B, Injection of MVFs into the dorsal skin flaps significantly improved flap survival when compared to PBS control animals at 14 days. Results are expressed as mean ± SEM; *statistical significance in comparison to PBS-treated animals (P < 0.05).

Fig. 3.

Laser Doppler measurements were recorded before surgery (Pre), after surgery (Post), 7 days postoperatively, and 14 days postoperatively. The values represented here were taken from all 3 regions of the flap. No significant differences were found when comparing MVF treated versus PBS treated at any time point for each location. Results are expressed as mean ± SEM.

Fig. 4.

A, Representative images of microvessel detection by fluorescein-labeled GSL I-isolectin B₄ immunofluorescence. Each image is from a different animal and shows the microvessel density in different regions of both PBS- and MVF-treated flaps. White scale bars represent 250 μm. B, Quantitation of the GSL I-isolectin B₄ immunofluorescence to determine microvessel density. Microvessel density is the number of GSL I-isolectin B₄ positive vessels per mm². A significant difference in microvessel density is shown between the regions but not between the treatments. Results are expressed as mean ± SEM; *statistical significance in comparison to other regions (P < 0.001).