Immunomodulation of Acellular Dermal Matrix Through Interleukin 4 Enhances Vascular Infiltration

Abstract

Background: Acellular dermal matrix (ADM) supported implant-based reconstruction remains the most commonly performed mode of reconstruction after breast cancer. Acellular dermal matrix clinical usage has reported benefits but requires rapid and efficient vascular and cellular incorporation into the recipient to have the best outcomes. Orderly transition from M1 to M2 macrophage phenotypic profile, coordinated in part by interleukin 4 (IL-4), is an important component of vascular stabilization and remodeling. Using the ADM substrate as a delivery device for immunomodulation of macrophage phenotype holds the potential to improve integration.

Methods: Interleukin 4 was adsorbed onto ADM samples and drug elution curves were measured. Next, experimental groups of 8 C57BL/6 mice had 5-mm ADM discs surgically placed in a dorsal window chamber with a vascularized skin flap on one side and a plastic cover slip on the other in a model of implant-based breast reconstruction. Group 1 consisted of IL-4 (5 μg) adsorbed into the ADM preoperatively and group 2 consisted of an untreated ADM control. Serial gross examinations were performed with histology at day 21 for markers of vascularization, mesenchymal cell infiltration, and macrophage lineage.

Results: Drug elution curves showed sustained IL-4 release for 10 days after adsorption. Serial gross examination showed similar rates of superficial vascular investment of the ADM beginning at the periphery by day 14 and increasing through day 21. Interleukin-4 treatment led to significantly increased CD31 staining of vascular endothelial cells within the ADM over the control group (P < 0.05) at 21 days. Although vimentin staining did not indicate a significant increase in fibroblasts overall, IL-4 did result in a significant increase in expression of α-smooth muscle actin. The expression of macrophage phenotype markers Arginase1 and iNOS present within the ADM were not significantly affected by IL-4 treatment at the day 21 time point.

Conclusions: Acellular dermal matrix has the potential to be used for immunomodulatory cytokine delivery during the timeframe of healing. Using implanted ADM as a delivery vehicle to drive IL-4 mediated angiogenesis and vascular remodeling significantly enhanced vascularity within the ADM substrate.

FIGURE 1.

Experimental schematic demonstrating the adsorption of IL-4 into ADM samples analyzed and placed into the wound in a murine dorsal skinfold model, where vascular investment was monitored for 21 days. At 21 days, samples were prepared and stained for cellular markers of wound healing.

FIGURE 2.

A, Release curve of IL-4–adsorbed ADM in nanogram per milliliter showing sustained release of IL-4 at a mean of 5.6 ng/mL over 10 days with tapering of IL-4 release over the subsequent 7 days (n = 4). B, Daily mean IL-4 release from IL-4–adsorbed ADM discs over 17 days expressed in nanogram per milliliter (n = 4).

FIGURE 3.

Serial gross 3× light microscopy examinations of 5-mm ADM specimens within the dorsal window chamber. Peripheral vessel can be noted penetrating the edges of the ADM at day 14 and increasing in surface area by day 21. Interleukin-4–treated ADM exhibited a more robust vascular response as compared with control samples through gross examination over 21 days.

FIGURE 4.

Hematoxylin and eosin–stained cross-section of ADM sample in the wound bed (5×, scale bar = 500 μm, zoom at 20×, scale bar = 100 μm). A, IL-4–treated ADM, B, Untreated control ADM. Cellular invasion that facilitates biointegration progresses from the base and edge of the ADM (arrows). At the interface of the ADM base and the wound bed, a highly cellular fibrous tissue is seen during healing (yellow line). The thickness of the capsule formation between the groups was not statistically different (404.5 ± 180.3 μm; +IL-4 vs 470.6 ± 208.3 μm; −IL-4).

FIGURE 5.

Number of CD31-positive endothelial cells by treatment group. Interleukin-4 elution ADM specimens demonstrated a significantly higher number of CD31-positive cells (39.1 ± 6.8 cells per 20× field of view at the base and 31.7 ± 9.0 at the edge) than control specimens (15.5 ± 7.2 at the base and 8.8 ± 6.7 at the edge) by t test (P < 0.05). (Scale bar = 100 μm).

FIGURE 6.

Percentage of field of view area stained positively for αSMA, a marker for smooth muscle cells and myofibroblasts. Interleukin-4 elution from ADM led to a significant increase in αSMA staining (9.5% ± 1.5% staining per 20× field of view at the base and 6.9% ± 1.7% at the edge) compared with untreated control ADM (2.9% ± 1.1% at the base and 3.8% ± 1.4% at the edge) by t test (P < 0.05). (Scale bar = 100 μm).

FIGURE 7.

Interleukin-4 treatment did not lead to a significant change in vimentin staining of fibroblasts as a percentage of field of view area. (Scale bar = 100 μm).

FIGURE 8.

Expression of iNOS and Arg1 marking M1 and M2 macrophage phenotype, respectively. By the 21-day histologic analysis time point, both the IL-4 and control groups demonstrated a slightly greater area percentage of Arg1 (9.8%–10.7%, A) staining positive per field as compared to iNOS (6.0–6.8%, B) staining positive per field, with no statistical differences.