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. 2008 Sep;149(1):47-56.
doi: 10.1016/j.jss.2007.12.788. Epub 2008 Jan 28.

Mold-shaped, Nanofiber Scaffold-Based Cartilage Engineering Using Human Mesenchymal Stem Cells and Bioreactor

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Free PMC article

Mold-shaped, Nanofiber Scaffold-Based Cartilage Engineering Using Human Mesenchymal Stem Cells and Bioreactor

Sasa Janjanin et al. J Surg Res. .
Free PMC article

Abstract

Background: Mesenchymal stem cell (MSC)-based tissue engineering is a promising future alternative to autologous cartilage grafting. This study evaluates the potential of using MSCs, seeded into electrospun, biodegradable polymeric nanofibrous scaffolds, to engineer cartilage with defined dimensions and shape, similar to grafts used for subcutaneous implantation in plastic and reconstructive surgery.

Materials and methods: Human bone marrow derived MSCs seeded onto nanofibrous scaffolds and placed in custom-designed molds were cultured for up to 42 days in bioreactors. Chondrogenesis was induced with either transforming growth factor-beta1 (TGF-beta1) alone or in combination with insulin-like growth factor-I (IGF-I).

Results: Constructs exhibited hyaline cartilage histology with desired thickness and shape as well as favorable tissue integrity and shape retention, suggesting the presence of elastic tissue. Time-dependent increase in cartilage matrix gene expression was seen in both types of culture: at Day 42, TGF-beta1/IGF-I treated cultures showed higher collagen Type 2 and aggrecan expression. Both culture conditions showed significant time-dependent increase in sulfated glycosaminoglycan and hydroxyproline contents. TGF-beta1/IGF-I-treated samples were significantly stiffer; with equilibrium compressive Young's modulus values reaching 17 kPa by Day 42.

Conclusions: The successful ex vivo development of geometrically defined cartilaginous construct using customized molding suggests the potential of cell-based cartilage tissue for reconstructive surgery.

Figures

Figure 1
Figure 1
Custom designed polypropylene molds with silicone sheet that defined dimensions and the shape of the tissue engineered tissue. One mold was designed to produce two pieces of tissue; each with dimensions of 10×10×2 mm. MSC-loaded nanofibrous scaffold can be visualized in the right chamber of the mold. Bar = 10 mm.
Figure 2
Figure 2
Spinner flask bioreactor culture system. Molds containing MSC-nanofiber composites are placed into the spinner flask, and maintained in a vertically suspended position due to the placement of stainless steel screws on one end (heavy) and polypropylene screws on the other end (light) of the mold. MSC-nanofiber constructs were cultured under chondrogenic conditions with addition of growth factors for 14, 28 and 42 Days.
Figure 3
Figure 3
Cartilaginous construct after 42 days in culture. Constructs were glossy in appearance, white, and resistant to physical deformation.
Figure 4
Figure 4
Cartilage matrix gene expression in engineered cartilage constructs analyzed by real-time, quantitative RT-PCR after Days 14, 28 and 42, respectively. MSC-nanofiber constructs were cultured in chondrogenic medium containing TGF-β1 alone or TGF-β1/IGF-I. Gene expression was analyzed by real-time RT-PCR for (A) aggrecan, (B) collagen type II, (C) collagen type I, and (D) elastin. mRNA values of each gene were normalized to those of the housekeeping gene, G3PDH. All values were mean ± SD. Data were analyzed using two-way ANOVA. Aggrecan, collagen type II, collagen type I, and elastin gene expression changed as a function of culture time (p<0.001 for aggrecan and collagen type II, p<0.05 for collagen type I, p<0.005 for elastin). Compared to TGF-β1 cultures, TGF-β1/IGF-I cultures did not show significant change of levels of mRNA expression of any of these markers (p>0.05).
Figure 5
Figure 5
DNA content of engineered cartilage constructs as a function of culture time. DNA content remained constant, and did not show significant increase over time or between the two growth factor culture conditions (TGF-β1 vs. TGF-β1/IGF-I; p>0.05). The average DNA content per single construct was 108.74±33.96 µg.
Figure 6
Figure 6
sGAG content of engineered cartilage constructs as a function of culture time and conditions. In both culture groups (TGF-β1 and TGF-β1/IGF-I), the amount of matrix sGAG changed significantly with time (p<0.01). There was no statistically significant difference in sGAG levels between the two growth factor conditions (p>0.05). Values are mean ± SD (n=6).
Figure 7
Figure 7
Collagen level in engineered cartilage constructs, estimated based on hydroxyproline content, as a function of culture time. In TGF-β1 and TGF-β1/IGF-I treated groups, hydroxyproline accumulation showed significant increase over time points (p<0.001). Values are mean ± SD (n=6). Data were analyzed using two-way ANOVA.
Figure 8
Figure 8
Mechanical properties of engineered cartilage constructs as a function of culture time. Equilibrium Young’s modulus of samples increased with culture time in both growth factor conditions (p<0.001). By Day 42, the TGF-β1/IGF-I cultures showed a significant increase compared to TGF-β1 cultures of Day 42 (p<0.05). Values are mean ± SD (n=4).
Figure 9
Figure 9
Histology of engineered cartilage constructs as a function of culture time. Both TGF-β1 and TGF-β1/IGF-I cultures display similar histology. (A–F) H&E staining. Regions of varying cell density are seen in early cultures (A, D: Day 14, and B, E: Day 28). A more uniform cell density is seen on Day 42 (C, F). Total cell number remains unchanged, as indicated by constant DNA content (see Figure 5). Bar = 100 µm. (G–N) Alcian blue staining. (G–L) High magnification showing increased Alcian blue staining as a function of culture time. Weak staining is seen on Day 14 (G, J). Cells become surrounded by sulfated proteoglycan-rich matrix beginning on Day 28 (H, K). By Day 42, samples from both medium conditions demonstrated extensive accumulation of sulfated proteoglycan-rich ECM. Bar = 100 µm. (M, N) Low magnification view of Day 28 constructs illustrating regional difference in staining intensity. Increased staining is seen at the edges (N), as compared to sections through the middle of the constructs (M). This pattern is not observed in Day 14 TGF-β1/IGF-I constructs (not shown). Bar = 500 µm.
Figure 9
Figure 9
Histology of engineered cartilage constructs as a function of culture time. Both TGF-β1 and TGF-β1/IGF-I cultures display similar histology. (A–F) H&E staining. Regions of varying cell density are seen in early cultures (A, D: Day 14, and B, E: Day 28). A more uniform cell density is seen on Day 42 (C, F). Total cell number remains unchanged, as indicated by constant DNA content (see Figure 5). Bar = 100 µm. (G–N) Alcian blue staining. (G–L) High magnification showing increased Alcian blue staining as a function of culture time. Weak staining is seen on Day 14 (G, J). Cells become surrounded by sulfated proteoglycan-rich matrix beginning on Day 28 (H, K). By Day 42, samples from both medium conditions demonstrated extensive accumulation of sulfated proteoglycan-rich ECM. Bar = 100 µm. (M, N) Low magnification view of Day 28 constructs illustrating regional difference in staining intensity. Increased staining is seen at the edges (N), as compared to sections through the middle of the constructs (M). This pattern is not observed in Day 14 TGF-β1/IGF-I constructs (not shown). Bar = 500 µm.
Figure 9
Figure 9
Histology of engineered cartilage constructs as a function of culture time. Both TGF-β1 and TGF-β1/IGF-I cultures display similar histology. (A–F) H&E staining. Regions of varying cell density are seen in early cultures (A, D: Day 14, and B, E: Day 28). A more uniform cell density is seen on Day 42 (C, F). Total cell number remains unchanged, as indicated by constant DNA content (see Figure 5). Bar = 100 µm. (G–N) Alcian blue staining. (G–L) High magnification showing increased Alcian blue staining as a function of culture time. Weak staining is seen on Day 14 (G, J). Cells become surrounded by sulfated proteoglycan-rich matrix beginning on Day 28 (H, K). By Day 42, samples from both medium conditions demonstrated extensive accumulation of sulfated proteoglycan-rich ECM. Bar = 100 µm. (M, N) Low magnification view of Day 28 constructs illustrating regional difference in staining intensity. Increased staining is seen at the edges (N), as compared to sections through the middle of the constructs (M). This pattern is not observed in Day 14 TGF-β1/IGF-I constructs (not shown). Bar = 500 µm.

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