A Graphene Composite Film Based Wearable Far‐Infrared Therapy Apparatus (GRAFT) for Effective Prevention of Postoperative Peritoneal Adhesion

Abstract Postoperative peritoneal adhesion (PPA) is the most frequent complication after abdominal surgery. Current anti‐adhesion strategies largely rely on the use of physical separating barriers creating an interface blocking peritoneal adhesion, which cannot reduce inflammation and suffers from limited anti‐adhesion efficacy with unwanted side effects. Here, by exploiting the alternative activated macrophages to alleviate inflammation in adhesion development, a flexible graphene‐composite‐film (F‐GCF) generating far‐infrared (FIR) irradiation that effectively modulates the macrophage phenotype toward the anti‐inflammatory M2 type, resulting in reduced PPA formation, is designed. The anti‐adhesion effect of the FIR generated by F‐GCF is determined in the rat abdominal wall abrasion‐cecum defect models, which exhibit reduced incidence and area of PPA by 67.0% and 92.1% after FIR treatment without skin damage, significantly superior to the clinically used chitosan hydrogel. Notably, within peritoneal macrophages, FIR reduces inflammation reaction and promotes tissue plasminogen activator (t‐PA) level via the polarization of peritoneal macrophages through upregulating Nr4a2 expression. To facilitate clinical use, a wirelessly controlled, wearable, F‐GCF‐based FIR therapy apparatus (GRAFT) is further developed and its remarkable anti‐adhesion ability in the porcine PPA model is revealed. Collectively, the physical, biochemical, and in vivo preclinical data provide compelling evidence demonstrating the clinical‐translational value of FIR in PPA prevention.


Figure S1 .
Figure S1.Temperature arising behaviors of the graphene film.a) The temperature-changing profile of a 30 cm × 35 cm F-GCF at different input voltages.b) The heating up curve of a 30 cm × 35 cm F-GCF during a 30-minute power supply (45 V).Aluminum foil blocking: the FIR was blocked using aluminum foil (~10 μm thickness).c) The measurement of the temperature of the pig's abdominal wall.d) The temperatures at different abdominal wall thicknesses with a film temperature of 42 °C (Troom = 28 °C).

Figure S2 .
Figure S2.The temperature of F-GCF under DC power supply for a duration of 20 days.a) The temperature of F-GCF on each day.Data are shown as mean ± SD; the temperature was measured 10 times per day.b) The representative thermal imaging graphs of F-GCF on the given day.

Figure S3 .
Figure S3.Flowchart and representative photographs of the establishment of rat cecum abrasion-abdominal wall defect model.

Figure S4 .
Figure S4.Evaluation of the safety of the FIR generated by F-GCF.a) After being treated by FIR for 7 days, the abdominal skins of rats were sampled and stained by H&E staining.For the "Heat only" group, the FIR was blocked using aluminum foil.Red arrows indicate the epidermis; Blue arrows indicate the follicles; Black arrows indicate collagen.Scar bars, 200 μm.b) Quantitation of the thickness of the epidermis.c) Numbers of follicles in abdomen skin.d) Quantitation of the thickness of the collagen in the abdomen skin.Data are shown as mean ± SD; six rats per group; one image per rat was randomly selected for quantification; p values were calculated using one-way ANOVA test.

Figure S5 .
Figure S5.The formation of cecum-abdominal adhesion of each rat on post-operation day 7.The white irregular dotted lines indicate the adhesion area; the white rectangle dotted lines indicate the non-adhesion abdominal wall.

Figure S6 .
Figure S6.The formation of cecum-abdominal adhesion after heat treatment on postoperation day 7. a) Schematic diagram of the experimental timeline.For the "heat only" (i.e., treated by thermal therapy alone) group, the film temperature was set to 42 °C; and an aluminum foil was covered on the film to block the FIR.b-d) The qualification of b) adhesion area, c) adhesion score, d) collagen deposition thickness on post-operation day 7. e) Representative photographs of the abdominal adhesions in heat-treated rats on post-operation day 7. CE: cecum; AD: adhesion; AW: abdominal wall.f) Representative H&E staining and Masson staining images of the adhesion tissues on post-operation day 7.The black dashed lines indicate the boundaries of adhesions.The images of control group are presented in the main text (Figure 2).Scale bars, 50 μm.Data are shown as mean ± SD; six rats per group; p values were calculated using two-tailed Student's t-test.

Figure S7 .
Figure S7.The formation of cecum-abdominal adhesions of the heat-treated rats on postoperation day 7.The images outlined by black dotted lines at the left are the enlargement of the adhesion area.AW: abdominal wall.

Figure S8 .
Figure S8.FIR treatment prevents the formation of long-term abdominal adhesion.a) Schematic diagram of the experimental timeline.b) Representative photographs of the abdominal adhesions in FIR-treated or untreated rats on post-operation day 70.CE: cecum; AD: adhesion; AW: abdominal wall.c) Representative H&E staining images of the adhesion tissues on post-operation day 70.The mesothelial monolayer was indicated by the blue arrows and the field is enlarged.The black dashed lines indicate the boundaries of adhesions.Scar bars, 100 μm in the original images, and 20 μm in the enlarged images.d) Representative Masson staining images of the adhesion tissues on post-operation day 70.Scale bars, 100 μm.e-h) The qualification of e) adhesion area, f) collagen deposition thickness, g) adhesion score, and h) the numbers of adhesive strips on post-operation day 70.i) The body weight of the rats.Data are shown as mean ± SD; six rats per group; p values were calculated using two-tailed Student's t-test.

Figure S9 .
Figure S9.The formation of cecum-abdominal adhesion of each rat on post-operation day 70.The images outlined by black dotted lines at the left are the enlargement of the adhesion area images or non-adhesion abdominal wall images.AW: abdominal wall.

Figure S10 .
Figure S10.The adhesive strips in the untreated rat and FIR-treated rat at post-operation day 70.

Figure S11 .
Figure S11.Representative images and quantification of immunohistochemical staining for CD68, MPO, TNF-α, and VEGF in adhesion tissues on post-operation day 7. a-d) Representative immunohistochemical staining images of a) macrophage marker CD68, b) neutrophil marker MPO, c) TNF-α, and d) VEGF in adhesion tissues (or abdominal wall injury site, for rats without adhesions) on post-operation day 7.The black dotted boxes in the upper panel were enlarged in the lower panel.The positive cells in corresponding groups were shown by black arrowheads.Scale bars, 100 μm.e-h) Quantification of e) CD68, f) MPO, g) TNF-α, and h) VEGF positive cells.Data are shown as mean ± SD; six rats per group; five images per rat were randomly selected for quantification; p values were calculated using twotailed Student's t-test.

Figure S12 .
Figure S12.Representative images and quantification of immunohistochemical staining of adhesion tissues on post-operation days 1 and 3. a) Representative images and b) quantification of macrophages (CD68 positive) in adhesion tissues (or abdominal wall injury site, for rats without adhesions) on post-operation days 1 and 3. c) Representative images and d) quantification of neutrophils (MPO positive) in adhesion tissues on post-operation day 1 and 3. e) Representative images and f) quantification of TNF-α in adhesion tissues on postoperation day 1 and 3. g) Representative images and h) quantification of VEGF in adhesion

Figure S13 .
Figure S13.FIR up-regulates the protein expression of Nr4a2.a and b) Western blot analysis of a) Nr4a1 and b) Nr4a2 protein expression in the LPS-treated rat primary peritoneal macrophages with or without FIR irradiation.The relative protein expression level was calculated as the gray intensity ratio of Nr4a1 or Nr4a2 versus GAPDH.c) The representative immunofluorescence staining images of Nr4a2 in the adhesion tissues.Scale bars, 50 μm.d) Quantification of the mean fluorescence intensity (MFI) of Nr4a2.e and f) Raw western blot images of e) Nr4a1 and f) Nr4a2 in the LPS-treated rat primary peritoneal macrophages with or without FIR irradiation.Data are shown as mean ± SD; 40 images from five rats (eight images per rat) in each group were randomly selected for quantification; p value was calculated using two-tailed Student's t-test.

Figure S14 .
Figure S14.siNr4a2 inhibits the expression of Nr4a2 in vitro.a) Western blot analysis of Nr4a2 protein expression in the siNr4a2 or negative control (NC) siRNA-transfected rat primary peritoneal macrophages.The western blot experiment was repeated three times.b) The representative immunofluorescence staining images of Nr4a2 in the siNr4a2 or negative control (NC) siRNA-transfected rat primary peritoneal macrophages.Scale bars, 10 μm.c) Quantification of the mean fluorescence intensity (MFI) of Nr4a2.Six images in each group were randomly selected for quantification.d) Raw western blot images of Nr4a2 protein

Figure S15 .
Figure S15.shNr4a2 lentivirus inhibits the expression of Nr4a2 in vivo.a) The representative immunofluorescence staining images of Nr4a2 in the adhesion tissues.Scale bars, 50 μm.b) Quantification of the mean fluorescence intensity (MFI) of Nr4a2.Data are shown as mean ± SD; 48 images from six rats (eight images per rat) in each group were randomly selected for quantification; p values were calculated using one-way ANOVA test.

Figure S16 .
Figure S16.The development of cecum-abdominal adhesion of each rat on day 7 after the indicated treatment.The images enclosed by white dashed lines on the left depict enlarged views of the adhesion area or non-adhesion abdominal wall images.

Figure S17 .
Figure S17.The thermal imaging graph (left), photograph (middle) of a wirelessly controlled, wearable FIR therapy apparatus (GRAFT), and the screenshot of the control software (right) when the treatment temperature was set to 42 °C.

Figure S18 .
Figure S18.Photograph of a pig with the wearable GRAFT.

Figure S19 .
Figure S19.Representative photographs of the establishment of porcine surgical adhesion model.a) A midline abdominal incision was made and the viscera were exposed; b) The contiguous small intestine (80 cm) was placed on dry gauze for 5 minutes; c) The small intestine was mechanically grazed with a surgical brush repeated 40 times until punctate bleeding appeared on the surface of the serosal layer; d,e) The fascia of the right abdominal wall peritoneum was excised to create a 6 cm × 4 cm peritoneal defect; f) The bleeding bowel segment and the damaged abdominal wall were sutured together.

Figure S20 .
Figure S20.The adhesion in each pig on post-operation day 7.The white irregular dashed lines indicate the adhesion area.The white rectangle dashed lines indicate the non-adhesion abdominal wall.