Catechol-rich gelatin microspheres as restorative medical implants intended for inhibiting seroma formation and promoting wound healing

Abstract

Seroma formation and poor wound healing are common complications of many surgeries that create anatomical dead space (i.e., mastectomy), often causing tissue infection and even necrosis. Although negative pressure drainage and tissue adhesives are investigated to alleviate fluid accumulation post-surgery, however, their therapeutic efficacy remains unsatisfactory in most cases. Herein, the catechol-rich chemically crosslinked gelatin microspheres (ca-CGMSs) have been developed as biodegradable reconstructive implants for preventing seroma formation and concurrently promoting subcutaneous wound healing. Compared with the most representative hydrogel adhesive, i.e. commercial porcine fibrin sealant (PFS), the loosely packed ca-CGMSs with diameters range from 50 to 350 μm, provide numerous cell-adhesive interfaces and interconnected macro-pores for enhanced cell adhesion, proliferation and migration. Subcutaneous embedding trials show the in situ swelling aggregation and wet tissue adhesion of ca-CGMSs as well as their capacity in recruiting autologous cells in rat mastectomy models. The trials in rabbit mastectomy models demonstrate that, compared with PFS gluing, the implanted dried ca-CGMSs not only significantly inhibit seroma formation, but also achieve enhanced wound healing by inducing the formation of vascularized neo-tissue. The ca-CGMSs show a great potential to be the next-generation of restorative materials for both preventing seroma formation and healing subcutaneous wounds.

Keywords: Catechol; Gelatin; Microsphere; Seroma; Tissue engineering; Wound healing.

Graphical abstract

Fig. 1

Schematic development of ca-CGMSs and their applications for both preventing seroma formation and healing subcutaneous wounds. The ca-CGMSs are prepared from the catechol-rich precursor solution with the water-in-oil emulsion method, followed by chemical crosslinking. After being transplanted into subcutaneous tissue defects, the ca-CGMSs not only form an aggregated construct and stick onto the wet tissue after absorbing seroma fluids, but also recruit autologous cells that achieve ingrowth into the tissue defect by migration and proliferation, realizing neo-tissue formation and wound healing.

Fig. 2

Preparation and characterization of ca-CGMSs. (A) Synthetic route of gel-dopa precursor and chemical crosslinking in formation of ca-CGMSs. (B) ¹H NMR spectra of gelatin, dopamine, and gel-dopa. (C) UV–Vis spectra of the solution (10 %, w/v) of gelatin and gel-dopa precursor, respectively. (D) Dried powder of ca-CGMSs. (E) SEM image of ca-CGMSs. (F) Equilibrium swelling ratios of ca-CGMSs fabricated with varied EDC/NHS concentrations and crosslinking times. (G) Optical image of the ca-CGMSs in PBS. (H) Diameter distribution of swollen ca-CGMSs in PBS. (I) Degradation profile of ca-CGMSs in trypsin solution at 37 °C.

Fig. 3

The microenvironments of ca-CGMSs favor cell survival, proliferation, and migration. (A) Live/dead analysis of L929 cells over culture time; living cells and dead cells are stained green and red, respectively; scale bar is 300 μm (B) Proliferation estimations of L929 cells on PFS and ca-CGMSs, respectively; statistical significance is indicated with ∗∗∗ (p ≤ 0.001) and ∗∗∗∗ (p ≤ 0.0001) (n = 5). (C) Crystal violet staining of the cells passing through (a) PFS or (b) ca-CGMSs into the lower transwell chamber; scale bar is 300 μm. (D) Quantification of the cells at the lower transwell chamber; statistical significance is indicated with ∗∗∗∗ (p ≤ 0.0001) (n = 5). (E) F-actin staining of L929 cells on PFS and ca-CGMSs, respectively, over culture time; scale bar is 300 μm.

Fig. 4

The dried ca-CGMSs inhibit seroma formation by absorbing fluid and adhering onto wet tissues, and meanwhile rapidly recruit endogenous cells. (A) The sequence of establishment of rat mastectomy model: (a) rats are placed in a supine position; (b) make a midline incision from jugular notch to xiphoid process; (c) remove muscles, fat, the upper mammary glands, and visible lymph nodes; (d) place the dried ca-CGMSs powder; (e) suture surgical areas. (B) Images of the ca-CGMSs after two days of transplantation; the aggregated ca-CGMSs constructs are highlighted with dashed circles that can withstand the weight of the isolated tissue. (C) Bright field image of isolated ca-CGMSs constructs; scale bar is 400 μm. (D) Fluorescent micrographs of the recruited cells in ca-CGMSs constructs after calcein-AM staining; scale bar is 300 μm. (E) F-actin staining of the isolated ca-CGMSs constructs; scale bar is 300 μm. (F) Photographs of the rabbit in steps of intra-operation and post-operation; the negative pressure drainage system is placed. (G) Volume of the collected seroma at each time point and total seroma volume of the three groups, respectively; statistical significance is indicated with ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), ∗∗∗ (p ≤ 0.001), and ∗∗∗∗ (p ≤ 0.0001) (n = 5).

Fig. 5

The ca-CGMSs promote the ingrowth of autologous cells into tissue defects. (A) F-actin staining of the tissue defects treated with PFS or ca-CGMSs, or not treated (Blank); scale bar is 300 μm. (B) Quantification of cell (nucleus) density in the tissue defects; statistical significance is indicated with ∗∗ (p ≤ 0.01), and ∗∗∗∗ (p ≤ 0.0001) (n = 5). (C) Schematic demonstration of cell recruitments in the three groups, respectively.

Fig. 6

Promotion of subcutaneous wound healing by ca-CGMSs in the rabbit mastectomy models. (A) Histological staining of formed neo-tissue with hematoxylin and eosin (H&E) and Masson Trichrome in three groups; ∗ indicates the sites of tissue defects; scale bar is 300 μm. Quantification of the deposited extracellular matrix (ECM) after (B) H&E and (C) Masson Trichrome staining, respectively; statistical significance is indicated with ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), ∗∗∗ (p ≤ 0.001), and ∗∗∗∗ (p ≤ 0.0001) (n = 5). (D) Sirius red staining of formed neo-tissue; ∗ indicates the sites of tissue defects; scale bar is 200 μm.

Fig. 7

The ca-CGMSs mediated neo-tissue formation in tissue defects in the rabbit mastectomy models. (A) The immunofluorescent stainings of (A) Col1 (the major ECM protein) and (B) vWF (one crucial angiogenic marker), respectively; ∗ indicates the sites of tissue defects; scale bar is 300 μm. Quantification of the deposited (C) Col1 and (D) vWF, respectively; statistical significance is indicated with ∗ (p ≤ 0.05), and ∗∗∗∗ (p ≤ 0.0001) (n = 5).

Fig. 8

The ca-CGMSs mediated the enhanced vascularization of the neo-tissue. (A) Immunohistochemistry staining of angiogenic markers, i.e., CD31, VEGF, and Hif-1α, respectively. ∗ indicates the sites of tissue defects; scale bar is 300 μm. (B) Quantification for the deposited CD31, VEGF, and Hif-1α, respectively; statistical significance is indicated with ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), ∗∗∗ (p ≤ 0.001), and ∗∗∗∗ (p ≤ 0.0001) (n = 5).