Decellularization of Wharton's Jelly Increases Its Bioactivity and Antibacterial Properties

Abstract

The field of regenerative medicine has recently seen an emerging trend toward decellularized extracellular matrix (ECM) as a biological scaffold for stem cell-delivery. Human umbilical cord represents a valuable opportunity from both technical and ethical point of view to obtain allogenic ECM. Herein, we established a protocol, allowing the full removal of cell membranes and nuclei moieties from Wharton's jelly (WJ) tissue. No alterations in the ECM components (i.e., collagen, GAG content, and growth factors), physical (i.e., porosity and swelling) and mechanical (i.e., linear tensile modulus) properties were noticed following WJ processing. Furthermore, no effect of the tissue processing on macromolecules and growth factors retention was observed, assuring thus a suitable bioactive matrix for cell maintenance upon recellularization. Based on the in vitro and in vivo biodegradability and stromal cell homing capabilities, decellularized WJ could provide an ideal substrate for stromal cells adhesion and colonization. Interestingly, the tissue processing increased the antibacterial and antiadhesive properties of WJ against Staphylococcus aureus and Staphylococcus epidermidis pathogens. Altogether, our results indicate that decellularized WJ matrix is able to limit Staphylococcus-related infections and to promote stromal cell homing, thus offering a versatile scaffold for tissue regenerative medicine.

Keywords: Wharton’s jelly; antibacterial; bioactivity; biocompatibility; decellularization; in vivo.

FIGURE 1

Structural characterization of d-WJ. (A): Macroscopical view of d-WJ and WJ control. (B): Nuclei moieties quantification in d-WJ and WJ, indicating a significant decrease in DNA content within d-WJ (red dashed line indicates the limit of detection of the used kit; n = 6, Mann & Whitney test). (C): Fluorescence microscopy observation of DAPI stained nuclei samples, indicating the absence of nuclear structures in d-WJ and the presence of nuclei in WJ samples as highlighted by white arrows (scale bars = 100 μm). (D): Hematoxylin-eosin-safran (HES, in upper left, black arrows highlighting stained nuclei), Masson’s trichrome (MT in upper right), Alcian Blue (AB at pH 1.5 in lower left and at pH 2.5 in lower right) of paraffin embedded d-WJ and WJ control (scale bars = 50 μm). (E): Biochemical assays for collagen, sulphated and non-sulphated glycosaminoglycans, indicating a preservation in ECM components within d-WJ (n = 4, Mann & Whitney test). (F): Infrared (IR) micro-spectroscopy spectra of d-WJ (grey) and WJ control (black) Spectra are normalized to one for the amide II band at 1,570 cm⁻¹. Insert represents second derivative spectra of d-WJ (grey) and WJ control (black) in the 1800–1,600 cm⁻¹ region.

FIGURE 2

Physical and mechanical characterization of d-WJ. (A): Scanning electron microscopy views at low magnification (in upper line) and high magnification (in lower line; scale bars = 20 and 100 μm, respectively). (B): Mercure intrusion porosity, (C,D): Two-photon excitation laser scanning confocal microscopy and second harmonic generation imaging (Scale bars = 25 μm) and signal quantification, respectively, indicating no alteration in collagen integrity within d-WJ (n = 4, Mann & Whitney test). (E): Equilibrium swelling ratio of d-WJ in PBS, showing a preservation in hydration capabilities of d-WJ (n = 4, Mann & Whitney test). (F): Mechanical testing photographs (in red is indicated the rupture at the fixation site) and (G): Linear elastic modulus of d-WJ (black bars) in dry and wet experimental conditions, showing no noticeable difference in d-WJ mechanical response versus WJ (white bars; n = 6, Mann & Whitney test).

FIGURE 3

Bioactivity of d-WJ matrix. (A): Mass spectrometry analysis of released macromolecules in d-WJ supernatant. (B): ELISA analysis of released growth factors, showing an increase growth factors release from d-WJ (n = 5, Mann & Whitney test).

FIGURE 4

Antibacterial properties of d-WJ. (A): Agar diffusion test, white arrows indicate zone of bacterial growth inhibition of S. aureus and S. epidermidis around d-WJ. (B): percentage of adhered bacteria on d-WJ matrix, and (C): Confocal laser microscopy visualization of Syto-9® labelled bacteria on d-WJ matrix (green color), indicating a significant decrease in bacteria adhesion on d-WJ (n = 9, Mann & Whitney test, scale bars = 25 μm).

FIGURE 5

In vitro biocompatibility of d-WJ. (A,B): Metabolic activities (black bars) and LDH release (blue bars) of WJ-SCs and fibroblasts, respectively in the presence of d-WJ and WJ control, indicating the absence of cytotoxic release of Triton X-100 from d-WJ (n = 6, Mann & Whitney test). (C): WJ-SCs proliferation assay and (D): DNA quantification of WJ-SCs cultured on d-WJ (black bars) and Bio-Gide® positive control (white bars), showing the absence of WJ-SCs proliferation on d-WJ (n = 6, Mann & Whitney test). (E, F): Cross sections of WJ-SCs cultured on d-WJ stained respectively with HES and DAPI. Arrows indicate cells within the d-WJ. (G): Confocal microscopy view of WJ-SCs cultured on d-WJ labelled with phalloidin (in green) and DAPI (in blue). Scale bars = 200 μm (E) and 25 μm (F,G).

FIGURE 6

In vivo Biocompatibility of d-WJ. (A): Masson’s Trichrome staining. (B, C): Hematoxylin-eosin-safran staining. Dashed delimitations indicate the implanted d-WJ. Picture in C represents a higher magnification of the yellow square in (B). White and black arrows indicate fibroblasts and blood vessels, respectively. Scale bars = 1 mm (A,B) and 20 μm (C).