Human Engineered Cartilage and Decellularized Matrix as an Alternative to Animal Osteoarthritis Model

Abstract

(1) Objective: to obtain a reproducible, robust, well-defined, and cost-affordable in vitro model of human cartilage degeneration, suitable for drug screening; (2) Methods: we proposed 3D models of engineered cartilage, considering two human chondrocyte sources (articular/nasal) and five culture methods (pellet, alginate beads, silk/alginate microcarriers, and decellularized cartilage). Engineered cartilages were treated with pro-inflammatory cytokine IL-1β to promote cartilage degradation; (3) Results: articular chondrocytes have been rejected since they exhibit low cellular doubling with respect to nasal cells, with longer culture time for cell expansion; furthermore, pellet and alginate bead cultures lead to insufficient cartilage matrix production. Decellularized cartilage resulted as good support for degeneration model, but long culture time and high cell amount are required to obtain the adequate scaffold colonization. Here, we proposed, for the first time, the combined use of decellularized cartilage, as aggrecanase substrate, with pellet, alginate beads, or silk/alginate microcarriers, as polymeric scaffolds for chondrocyte cultures. This approach enables the development of suitable models of cartilaginous pathology. The results obtained after cryopreservation also demonstrated that beads and microcarriers are able to preserve chondrocyte functionality and metabolic activity; (4) Conclusions: alginate and silk/alginate-based scaffolds can be easily produced and cryopreserved to obtain a cost-affordable and ready-to-use polymer-based product for the subsequent screening of anti-inflammatory drugs for cartilage diseases.

Keywords: alginate; beads; decellularized cartilage matrix; human chondrocytes; microcarrier; osteoarthritis; pellet; silk fibroin.

Figure 1

Schematic representation of study’s experimental design. Five 3D models were proposed considering the type of scaffolds, the number of cells/test, and the in vitro culture time as process parameters.

Figure 2

Optical microscope images of two different cell lines of articular (ACs) and nasal septal (NCs) chondrocytes cultured in monolayer condition until sub-confluence for eight and 14 days, respectively (25× magnification). Cells appeared as spindle-shaped without morphological differences.

Figure 3

Mean values and standard deviation of µg GAG/bead, produced by encapsulated articular (ACs) and nasal (NCs) chondrocytes after 1, 2, 4, 5, and 7 weeks of culture. At each considered time, three beads were analyzed to determine the GAG production; overall, six cell lines were considered for ACs and six cell lines were considered for NCs. Unloaded beads were considered as the negative control (experimental sampe size = 90).

Figure 4

Relative cell metabolic activity (percentage) of chondrocytes for each 3D model before (grey bars) and after (white bars) stimulus with pro-inflammatory cytokine IL-1β (48 h). Mean values obtained from three cell lines ± standard deviations. Each condition was tested in triplicate (experimental sample size = 90).

Figure 5

Mean values and standard deviation of µg GAG/mL released by NCs before and after the treatment with IL-1β, for each 3D model.

Figure 6

Live (green) and dead (red) staining of encapsulated NCs (beads model) after cryopreservation. Magnification at 5× (A,B) and 10× (C,D). Relative cell metabolic activity % (E), expressed as mean values ± standard deviation, of encapsulated NCs before cryopreservation (control) and after the thawing process (t0-2-4-9 days). Different letters indicate significant differences (p < 0.05). Overall we considered three bead batches produced from three nasal chondrocyte lines, and, for each considered time, three beads were analyzed (experimental sample size n = 45).

Figure 7

Formazan staining for viable cells on silk/alginate microcarriers, before cryopreservation (A) and seven days after thawing (B). Magnification of 5×. Relative cell metabolic activity % (C), expressed as mean values ± standard deviation of encapsulated chondrocytes before cryopreservation (control) and after thawing process (t0-2-4-9 days). Different letters indicate significant differences (p < 0.05). Overall we considered three bead batches, produced from three nasal chondrocyte lines and, for each considered time, three beads were analyzed (experimental sample size n = 45).

Figure 8

Histological evaluation of encapsulated NCs (beads model) after 35 days of culture post cryopreservation: Hematoxylin-eosin staining (a,b) and immunostaining for type II collagen (the phenotypic marker for chondrocytes) (c,d).