Zwitterion nanocomposite hydrogels with bioactivity and anti-adhesion properties for rapid prevention of postoperative and recurrent adhesion

Abstract

Postoperative adhesions (PAs) are a common complication after intraperitoneal surgery. Hydrogels are a physical barrier that prevents peritoneal adhesions, but their efficacy is still controversial. In this study, Laponites, a layered two-dimensional nanoscale, is incorporated into zwitterionic hydrogel (pSBLA) to enhanced biocompatibility and bioactivity to develop a nanocomposite for rapid prevention of postoperative and recurrent adhesion. The anisotropic distribution of charges in laponites results in strong hydrogen bonding and electrostatic repulsion in aqueous solutions and enables hydrogen bonding between amphiphilic ions, thereby enhancing the mechanical properties of hydrogels. The pSBLA hydrogels also possess a series of characters for an ideal anti-adhesion material, including resistance to adhesion against fibrinogen, proteins as well as cells. The mechanism underlying the extraordinary hydration of pSBLA is elucidated in this study using molecular dynamic simulations. In addition, pSBLA hydrogel is shown to represent a major advancement in anti-adhesion efficacy by completely and reliably preventing postoperative and recurrent adhesions after adhesiolysis in rat models. Furthermore, mechanistic explorations revealed that pSBLA hydrogel inhibits inflammatory responses and resists fibrosis by regulating the transforming growth factor-β/Smad signal pathway. Therefore, the pSBLA hydrogel has considerable potential for preventing post-operative adhesions in clinical settings.

Keywords: Nanosilicate; Nonfouling performances; Postoperative adhesions; Zwitterionic hydrogels.

Graphical abstract

Scheme 1

(a) Schematic illustration of the formation of the pSBLA hydrogel. (b) Cecum-abdominal wall adhesion model establishment and different effects with or without the treatment of pSBLA2 hydrogel. (c) Schematic depicting the process and mechanism of the hydrogel for inhibiting postoperative abdominal adhesion. (Created with BioRender.com).

Fig. 1

Characterization of pSBLA hydrogels. (a) The optical photograph of pSBMA and pSBLA2 hydrogel in synthesis vial. (b) The interior structure of the dry materials. Scale bar = 50 μm. (c) FT-IR spectra and (d) XPS diagram of pSBLA2, pSBMA, Laponites. (e) TGA curve of Laponties, pSBMA, and pSBLA2 hydrogels. (f) The rheological characterization of hydrogels in frequency-sweep (0.1–10 Hz, strain of 1 %), (g) in time-sweep curves (10 min, 1 Hz, strain of 1 %, 37 °C), and (h) viscosity curves with a low strain at 1 %. (i) The water lose rate of pSBMA, pSBLA0.5, pSBLA1, and pSBLA2.

Fig. 2

The biocompatibility of pSBLA hydrogels in vitro. The images (a) and quantitative statistics (b) of blood incubated with hydrogels. (c) Live/dead cell viability assay. NIH/3T3 fibroblast cells were incubated with both the hydrogel extract for 24 h and 72 h. In the assay, live cells stain green and dead cells stain red, as viewed with a fluorescent microscope. Scale bar = 100 μm. CCK-8 assay for evaluation of cytotoxicity of different samples for 24 h (d) and 72 h (e). (Data presented as mean ± SD, n = 3, ∗∗p < 0.001, compared with the control group). (f) In vitro scratch assay, scale bar = 100 μm. Quantified data of the migration rate (mobility ratio) after the treatments with the different samples for 12 h (g) and 24 h (h) (n = 3, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05, compared with the control group). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The nonfouling performances of pSBLA hydrogels in vitro. The relative adhesion behaviors of the (a) fibrinogen, (b) BSA, and (c) platelets on tissue culture polystyrene (TCPS) and different hydrogel. (n = 3, ∗∗∗∗p < 0.0001). (d) Fluorescent images a of NIH/3T3 cells cultured for 24 h on different hydrogel surfaces. Scar bar = 100 μm.

Fig. 4

The mechanism of the protein and cell resistance was investigated at molecular level by MD simulation. (a) The probability distributions for the number of hydrogen bonds. (b) Autocorrelation function for τ_HB, C_HB (t), for water-SBMA sulfonyl oxygen, water-pSBMA sulfonyl oxygen, water-OEG hydroxyl oxygen, and water-water hydrogen bonds. (c) RDF of water oxygen with respect to one sulfonyl O, N, and S atoms of SBMA. (d) Snapshot of aqueous SBMA with opaque water corresponding to the main peak in the RDF of water oxygen with respect to O (high opacity), N, S atoms of SBMA, respectively. (e) Snapshot of pSBMA in aqueous solution, highlighting the tightly bound waters near two of the SBMA headgroup.

Fig. 5

Evaluation of the pSBLA hydrogel for preventing postsurgical abdominal adhesion using a rat sidewall defect-cecum abrasion adhesion model. (a) Schematic of the procedure schedule. (b) Photographs of peritoneal adhesions in the model, HA hydrogel, and pSBLA2 hydrogel group on postoperative day 7 and 14. (c) Adhesion score of the different degrees in each group on the predetermined time points. (n = 4, ∗∗∗∗p < 0.0001). Representative H&E and Masson trichrome staining of adhesive tissues from different groups on day 7 (d) and day 14 (e) after first surgery. CE: cecal mucosa; AW: abdominal wall, Scale bar = 200 μm.

Fig. 6

Expression levels of TNF-α and IL-1β in injured tissues of different groups. (a, b) Immumohistochemical staining of injured tissues after 7 day and 14 day (scale bar = 200 μm). Quantitative analyses of (c) IL-1β and (d) TNF-α (n = 3, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). The mRNA levels of (e) IL-1β and (f) TNF-α in injured tissues from different groups. (g, h) Effect of pSBLA2 hydrogel on the expression of cytokine IL-1β and TNF-α in tissue on day 7,14 post-operation (n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, compared with control group).

Fig. 7

Expression levels of TGF-β1 in injured tissues of different groups. (a) Secretion levels of TGF-β1 from tissues after treatment with different materials on day 7 and day14 by ELISA (n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). (b) mRNA levels of representative TGF-β1 in tissues after different treatment (n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). (c) Immumohistochemical staining and (d) quantitative analyses of injured tissues after 7 day and 14 days (scale bar = 200 μm, n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (e) Expression levels and (f, g) quantitative analyses of TGF-β1 markers in tissue via WB assay on day 7,14 post-operation.

Fig. 8

(a, b) Representative images of immunofluorescence staining of Smad 2/3 and Smad 7 on day 7 and day 14. (c, d) Mean fluorescence intensity (MFI) of Smad2/3 and Smad7 on day 7 and day 14, respectively. (n = 3, ∗∗∗∗p < 0.0001). (e–g) Western blot analysis and quantitative of protein expressions (Smad2/3 and Smad7) on day 7, 14. (H) Molecular mechanism of pSBLA2 hydrogel in preventing postoperative abdominal adhesions. (Created with BioRender.com).

Fig. 9

Evaluation of the pSBLA hydrogel for preventing postsurgical recurrent adhesion using a rat sidewall defect-cecum abrasion adhesion model. (a) Schematic of the procedure schedule. (b) Photographs of peritoneal adhesions in the model, HA hydrogel, and pSBLA2 hydrogel group on day 7 after adhesiolysis. (c) The relative body weight and (d) adhesion score of the different degrees in each group on day 7 after adhesiolysis. (n = 4, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (e, f) H&E and Masson trichrome staining of adhesive tissues from different groups on day 7 after adhesiolysis. Scale bars = 200 μm. CE: cecal mucosa; AW: abdominal wall.