Combination of Collagen-Based Scaffold and Bioactive Factors Induces Adipose-Derived Mesenchymal Stem Cells Chondrogenic Differentiation In vitro

Abstract

Recently, multipotent mesenchymal stem cells (MSCs) have attracted much attention in the field of regenerative medicine due to their ability to give rise to different cell types, including chondrocytes. Damaged articular cartilage repair is one of the most challenging issues for regenerative medicine, due to the intrinsic limited capability of cartilage to heal because of its avascular nature. While surgical approaches like chondral autografts and allografts provide symptoms and function improvement only for a short period, MSC based stimulation therapies, like microfracture surgery or autologous matrix-induced chondrogenesis demonstrate to be more effective. The use of adult chondrocytes, which are the main cellular constituent of cartilage, in medical practice, is indeed limited due to their instability in monolayer culture and difficulty to collect donor tissue (articular and nasal cartilage). The most recent cartilage engineering approaches combine cells, biomaterial scaffold and bioactive factors to promote functional tissue replacements. Many recent evidences demonstrate that scaffolds providing specific microenvironmental conditions can promote MSCs differentiation toward a functional phenotype. In the present work, the chondrogenic potential of a new Collagen I based 3D scaffold has been assessed in vitro, in combination with human adipose-derived MSCs which possess a higher chondrogenic potential compared to MSCs isolated from other tissues. Our data indicate that the scaffold was able to promote the early stages of chondrogenic commitment and that supplementation of specific soluble factors was able to induce the complete differentiation of MSCs in chondrocytes as demonstrated by the appearance of cartilage distinctive markers (Sox 9, Aggrecan, Matrilin-1, and Collagen II), as well as by the cartilage-specific Alcian Blue staining and by the acquisition of typical cellular morphology. Such evidences suggest that the investigated scaffold formulation could be suitable for the production of medical devices that can be beneficial in the field of articular cartilage engineering, thus improving the efficacy and durability of the current therapeutic options.

Keywords: 3D scaffolds; cartilage repair; chondrogenic differentiation; mesenchymal stem cells; regenerative medicine.

Figure 1

SEM images of collagen scaffold at (A) 30x, (B) 50x, and (C) 150x magnifications. (D) Graphical representation of pore size (μm) frequency throughout the scaffold. (E) Graphical representation of collagen scaffold swelling behavior relative to the modification of diameter and thickness after soaking in PBS solution. Blue columns, dry samples before soaking; red columns, wet samples after soaking.

Figure 2

Immunofluorescent (A) and flow cytometry analyses (B) of negative (CD31, CD34, CD45, and GlycoforinA) and positive (CD73, CD90, CD105, and CD271) mesenchymal stem cells surface markers (red and green stains). Nuclei are labeled in blue. Power magnification: 20x. Scale bar: 100 μm.

Figure 3

(A) Haematoxylin-Eosin staining of hADSC cultured on scaffolds either in absence (expansion medium) or presence (chondro medium) of chondrogenic inducing factors in culture media, after 1 (A,B), 2 (C,D), 4 (E,F), and 8 (G,H) weeks of growth in culture, magnification 20x. (I–L) higher power magnifications of the squared area, respectively, in (B,D,F,H). The arrows in (K) indicate the presence of pericellular lacunae resembling chondrocytes.

Figure 4

Alcian Blue staining of hADSC cultured on scaffolds either in absence (expansion medium) or presence (chondro medium) of chondrogenic inducing factors in culture media, after 1 (A,B), 2 (C,D), 4 (E,F), and 8 (G,H) weeks of growth in culture, magnification 20x. (I–L) higher power magnifications of the squared area shown, respectively, in (B,D,F,H). The arrows in (K) indicate the presence of pericellular lacunae resembling chondrocytes.

Figure 5

Immunofluorescent analysis of representative chondrogenic markers: (A–I) Sox9, (J–R) type II Collagen, performed on hADSCs and hADSCs cultured on scaffolds either in expansion (G–I,P–R) or in chondrogenic (A–F,J–O) medium, at different time points (1, 2, and 4 weeks), magnification 20x. (X) Average cellular positivity for chondrogenic markers in both expansion (dark bars) and chondrogenic medium (dark and medium bars). Percentage of Sox9 and Collagen II (X) are calculated on the total number of DAPI stained cells in the investigated fields. Two-way ANOVA p values are reported. Symbols above bars indicate statistically significant differences (p < 0.05) in the Tukey HSD post-hoc tests: 1 indicates differences with the 1 week group (same medium), 2 indicates differences with the 2 weeks group (same medium), 3 indicates differences with the 4 weeks group (same medium), 4 indicates differences with the 8 weeks group (same medium), ^* indicates differences with scaffold expansion medium group (same time-points), # indicates differences with scaffold chondrogenic medium group (same timepoints) and % indicates differences with pellet group (same timepoints). Average cellular positivity for chondrogenic markers at 2 (dark bars) and 4 weeks (light bars). Three independently isolated hADSC samples have been used for each time-point.

Figure 6

Immunofluorescent analysis of representative chondrogenic markers: (A–I) Aggrecan, (J–R) Matrilin, performed on hADSCs and hADSCs cultured on scaffolds either in expansion (G–I,P–R) or in chondrogenic (A–F,J–O) medium, at different time points (1, 2, and 4 weeks), magnification 20x. (X) Average cellular positivity for chondrogenic markers in both expansion (dark bars) and chondrogenic medium (dark and medium bars). Percentage of Aggrecan and Matrilin (X) are calculated on the total number of DAPI stained cells in the investigated fields. Two-way ANOVA p values are reported. Symbols above bars indicate statistically significant differences (p < 0.05) in the Tukey HSD post-hoc tests: 1 indicates differences with the 1 week group (same medium), 2 indicates differences with the 2 weeks group (same medium), 3 indicates differences with the 4 weeks group (same medium), 4 indicates differences with the 8 weeks group (same medium), ^* indicates differences with scaffold expansion medium group (same time-points), # indicates differences with scaffold chondrogenic medium group (same timepoints) and % indicates differences with pellet group (same timepoints). Average cellular positivity for chondrogenic markers at 2 (dark bars) and 4 weeks (light bars). Three independently isolated hADSC samples have been used for each time-point.