3D Porous Gelatin/PVA Hydrogel as Meniscus Substitute Using Alginate Micro-Particles as Porogens

Abstract

One of the current major challenges in orthopedic surgery is the treatment of meniscal lesions. Some of the main issues include mechanical consistency of meniscal implants, besides their fixation methods and integration with the host tissues. To tackle these aspects we realized a micro-porous, gelatin/polyvinyl alcohol (PVA)-based hydrogel to approach the high percentage of water present in the native meniscal tissue, recapitulating its biomechanical features, and, at the same time, realizing a porous implant, permissive to cell infiltration and tissue integration. In particular, we adopted aerodynamically-assisted jetting technology to realize sodium alginate micro-particles with controlled dimensions to be used as porogens. The porous hydrogels were realized through freezing-thawing cycles, followed by alginate particles leaching. Composite hydrogels showed a high porosity (74%) and an open porous structure, while preserving the elasticity behavior (E = 0.25 MPa) and high water content, typical of PVA-based hydrogels. The ex vivo animal model validation proved that the addition of gelatin, combined with the micro-porosity of the hydrogel, enhanced implant integration with the host tissue, allowing penetration of host cells within the construct boundaries. Altogether, these results show that the combined use of a water-insoluble micro-porogen and gelatin, as a bioactive agent, allowed the realization of a porous composite PVA-based hydrogel to be envisaged as a potential meniscal substitute.

Keywords: alginate micro-particles; ex vivo culture; gelatin; meniscus; polyvinyl alcohol; porogen leaching; porous hydrogel.

Figure 1

Optical image of the alginate microspheres (a) and the Probability Density Function of their diameter distribution (b).

Figure 2

Schematic representation of the fabrication process of gelatin/polyvinyl alcohol (PVA) porous hydrogels.

Figure 3

Optical images depict the micro-porosity of the hydrogels after 2 days (a), 5 days (b) and 10 days (c) of incubation in Ethylenediaminetetraacetic acid (2,2′,2″,2′′′-(Ethane-1,2-diyldinitrilo) tetraacetic acid; EDTA) solution. Red arrows indicate alginate residuals. Scale bar: 100 micron.

Figure 3

Optical images depict the micro-porosity of the hydrogels after 2 days (a), 5 days (b) and 10 days (c) of incubation in Ethylenediaminetetraacetic acid (2,2′,2″,2′′′-(Ethane-1,2-diyldinitrilo) tetraacetic acid; EDTA) solution. Red arrows indicate alginate residuals. Scale bar: 100 micron.

Figure 4

FT-IR spectra of PVA, gelatin and composite gelatin/PVA hydrogels.

Figure 5

Swelling ratio of both porous and non-porous hydrogels after various soaking time in phosphate-buffered saline (PBS)-based solution.

Figure 6

Cell viability of 3T3s after 24 h of culture on PVA (a) and gelatin/PVA (b) hydrogel film. Green spots are alive cells, red spots are dead ones. DAPI staining on histological sections representing nuclei (blue staining) of 3T3s adhering over the 3D porous PVA hydrogel (c), and composite porous gelatin/PVA hydrogel (d).

Figure 7

Ex Vivo culture model by bovine meniscus (a). Histology images (H&E) representing the junctions between the meniscal explants and the gelatin/PVA porous hydrogel (b), PVA porous hydrogel (c), non-porous gelatin/PVA hydrogel (d), sham (e) after 10 days of ex vivo culture. Meniscal explants (m), hydrogels (h), and the sham (sh) are indicated in the figure.

Figure 8

DAPI staining on histological sections representing nuclei (indicated by white arrows) of meniscus cells at the junctions level between the meniscal explants and the gelatin/PVA porous hydrogel (a), PVA porous hydrogel (b), non-porous gelatin/PVA hydrogel (c), sham (d) after 10 days of ex vivo culture. Meniscal explants (m), hydrogels (h) and the sham (sh) are indicated in the figure.