Dedifferentiated Chondrocytes in Composite Microfibers As Tool for Cartilage Repair

Abstract

Tissue engineering (TE) approaches using biomaterials have gain important roles in the regeneration of cartilage. This paper describes the production by microfluidics of alginate-based microfibers containing both extracellular matrix (ECM)-derived biomaterials and chondrocytes. As ECM components gelatin or decellularized urinary bladder matrix (UBM) were investigated. The effectiveness of the composite microfibers has been tested to modulate the behavior and redifferentiation of dedifferentiated chondrocytes. The complete redifferentiation, at the single-cell level, of the chondrocytes, without cell aggregate formation, was observed after 14 days of cell culture. Specific chondrogenic markers and high cellular secretory activity was observed in embedded cells. Notably, no sign of collagen type 10 deposition was determined. The obtained data suggest that dedifferentiated chondrocytes regain a functional chondrocyte phenotype when embedded in appropriate 3D scaffold based on alginate plus gelatin or UBM. The proposed scaffolds are indeed valuable to form a cellular microenvironment mimicking the in vivo ECM, opening the way to their use in cartilage TE.

Keywords: cartilage tissue engineering; chondrocytes; extracellular matrix-derived biomaterials; gene expression; microfibers.

Figure 1

Production and characterization of composite microfibers. (A) Schematic representation of the snake micromixing microfluidic device employed for the production of composite microfibers. The device presents two inlets where the alginate suspension containing cells or gelatin or UBM were delivered through the micromixing snake geometry channel and a 700-µm outlet tube (#T3) into a BaCl₂ solution in order to obtain alginate (Af), alginate plus gelatin (AGf), or alginate plus UBM (AUBMf) microfibers. (B) Photomicrographs of Af, AGf, and AUBMf obtained at different pumping rates (from 0.5 to 1.5 mL/min). Arrows indicate the presence in AUBMf of UBM particles, in form of flat flakes. Bar corresponds to 500 µm. (C) Effect of the pumping rate on the dimension of the produced microfibers.

Figure 2

Distribution of gelatin and UBM in composite microfibers. Different amounts of gelatin (1.125, 2.25, and 4.5% w/v) or UBM (0.1, 0.5, and 1% w/v) were added to the alginate solution, respectively. Microfibers containing different amount gelatin/UBM were stained with Coomassie Blue Brilliant. The presence and distribution of UBM particles are indicated by arrows. Bar corresponds to 1 mm in lower magnification photomicrographs and to 400 µm in higher magnification images.

Figure 3

Schematic representation of the experimental approach. Human chondrocytes were isolated from nasal septum biopsy, expanded, and dedifferentiated up to the sixth culture passage (P6). Cells were then embedded in Af, AGf, and AUBMf and subjected to the indicated analysis at days 7 and 14. Dedifferentiation process from passage 0 (P0) to passage 6 (P6) has been monitored: protein expression of cartilage-related genes (Col2A1, Aggrecan, and Sox9), and Col1A1 was investigated by immunocytochemical analysis. Alcian Blue staining for sulfate glycosaminoglycans (GAGs) detection is also reported. Cell morphology was evaluated by hematoxylin staining. Representative optical photomicrographs are reported. Pictures of at least four random fields of three replicates were captured for densitometric analysis using ImageJ software. Data are presented as means of pixels per 100 cells ±SEM (*p ≤ 0.05). Cell viability of embedded cells has also been investigated, and optical and fluorescence photomicrographs after double staining with Calcein-AM/propidium iodide at days 7 and 14 of culture in basal medium are reported. The green fluorescence indicates the presence of calcein-labeled live cells, while propidium iodide-labeled dead cells are revealed by red fluorescence. Merged photomicrographs are reported.

Figure 4

Properties of the embedded cells: presence of secretory vesicles and metachromatic areas in the extracellular matrix (ECM). Af, AGf, and AUBMf embedded cells at days 7 and 14 of culture were stained with toluidine blue. Representative photomicrographs showed the presence of ECM deposition in the pericellular space of the single cells (as clearly evidenced in the higher magnification images) and the presence of metachromatic area (pink). Black arrows: single or UBM-attached cell. Bar corresponds to 50 µm for photomicrographs (A,D,G,J,M,P), to 25 µm for photomicrographs (B,E,H,K,N,Q), and to 10 µm for photomicrographs (C,F,I,L,O,R).

Figure 5

Chondrogenic properties of Af, AGf, and AUBMf embedded cells: transmission electronic microscope ultrastructural analysis at day 14 of culture. Lower magnification photomicrographs showed the ultrastructure of an embedded redifferentiated chondrocyte in Af (A), AGf (B), and AUBMf (C). In the images at higher magnification, red arrows indicated the presence of ECM dense material and collagen fibers with their typical banding pattern in the pericellular space (D,E) and evidenced the ECM-containing vesicles release from embedded cells (F,G). Bar corresponds to 2.6 µm in a-c, 1 µm in (D–F) and 0.4 µm in grams. C, cell; U, UBM.

Figure 6

Chondrogenic properties of Af, AGf, and AUBMf embedded cells: analysis of cartilage-specific markers. Af, AGf, and AUBMf embedded redifferentiated chondrocytes or chondrocyte micromasses (MM) cultured for 7 or 14 days were compared for chondrogenic capacity by analyzing the expression of cartilage markers. (A) The expression of Col2A1, Col1A1, and aggrecan was evaluated by RT-qPCR. Values obtained from freshly isolated (P0) and dedifferentiated chondrocytes (P6) are also included. Results were calculated using 2^−ΔΔCt method, and data are presented as fold change means respect to MM day 7. (B) Quantification of Col2a1/Col1a1 ratio, Col10A1 expression, and GAG content for each experimental condition are reported. Col10a1 expression was assessed by RT-qPCR and resulted not detectable (ND) in all tested conditions. GAG content was quantified by DMMB staining on cellular lysates, and values are reported as µg GAG/ng DNA. All data are presented as means ± SEM of three independent experiments performed on chondrocytes from four different donors (n = 4). Statistical analysis was performed all conditions versus MM day 7 (*p < 0.05) or versus MM day 14 (^Δp < 0.05).

Figure 7

Behavior of the embedded cells after they are recovered from the microfibers. Redifferentiated chondrocytes were recovered from microfibers after 14 days of culture, reseeded, grown up to 7 days as monolayered culture in standard medium without adding chondrogenic inducers, and compared with P6 dedifferentiated chondrocytes. The cells were stained by Alcian blue for sulfated GAGs and immunostained for Col2A1 after growing in monolayer for 16 h, 72 h, or 7 days. Optical photomicrographs indicate the maintenance of acquired chondrogenic properties. Bar corresponds to 50 and 100 µm for the insets.

Figure 8

Cryopreservation of Af, AGf, and AUBMf embedded cells. Af, AGf, and AUBMf embedded redifferentiated chondrocytes were frozen, stored in liquid nitrogen, thawed, and assessed for cell morphology, viability, and chondrogenic properties. Optical images of microfibers show the rounded shape retention by the thawed embedded cells (particularly evident at higher magnification: see white arrows). Fluorescence merged images of the Calcein-AM (green)/propidium iodide (red) double staining demonstrated the high cell viability. The presence of GAGs and Col2A1 was detected by Alcian Blue staining and immunocytochemical analysis on the thawed embedded cells recovered from microfibers and grown in monolayer for 24 h. Bar corresponds to: 300 µm in lower magnification images of morphology and viability, 60 µm in higher magnification, and 50 µm in Alcian Blue staining and Col2A1 immunocytochemical analysis.