A comparison of the influence of material on in vitro cartilage tissue engineering with PCL, PGS, and POC 3D scaffold architecture seeded with chondrocytes

Abstract

The goal of this study was to determine material effects on cartilage regeneration for scaffolds with the same controlled architecture. The 3D polycaprolactone (PCL), poly (glycerol sebacate) (PGS), and poly (1,8 octanediol-co-citrate) (POC) scaffolds of the same design were physically characterized and tissue regeneration in terms of cell phenotype, cellular proliferation and differentiation, and matrix production were compared to find which material would be most optimal for cartilage regeneration in vitro. POC provided the best support for cartilage regeneration in terms of tissue ingrowth, matrix production, and relative mRNA expressions for chondrocyte differentiation (Col2/Col1). PGS was seen as the least favorable material for cartilage based on its relatively high de-differentiation (Col1), hypertrophic mRNA expression (Col10) and high matrix degradation (MMP13, MMP3) results. PCL still provided microenvironments suitable for cells to be active yet it seemed to cause de-differentiation (Col1) of chondrocytes inside the scaffold while many cells migrated out, growing cartilage outside the scaffold.

Figure 1

(A) Top view of MicroCT image of a scaffold (B) a digital picture of a POC scaffold (C) Side view of MicroCT image of a scaffold (D) Isosurfaced 3D MicroCT image of a scaffold

Figure 2

Digital pictures of three different material scaffolds with tissues grown for 4 weeks.

Figure 3

(A) Amount of DNAs per construct at 4 weeks for different materials (PCL_in: tissues inside PCL scaffolds only, PCL out: excessive outer layers removed from PCL scaffolds, PCL total = PCL_in + PCL_out) (Annotations ‘a’, ‘b’, ‘c’ shown in the graphs are statistically significant each other; PCL_in, POC are significant to all other groups, PGS are significant to PCL in and POC only) (B) Changes in DNA content of chondrocytes for different materials over time is measured by amount of DNAs per scaffold suggesting some possible cell migration (especially for PCL) and exterior tissue growth. (Asterisk represents statistical significance. p≤0.05, N=6) (C) Matrix production per scaffold is quantified by amount of sGAG per construct for different materials. (PCL_in, POC are significant to all other groups, PGS are significant to PCL in and POC, p≤0.05, N=6)

Figure 4

Relative mRNA expression comparison for proteins among different materials. (PCL = PCL inner tissues only) (Annotations ‘a’, ‘b’, ‘c’ shown in the graphs are statistically significant each other. N=6, p≤0.05)

Figure 5

Safranin-O/Fast-Green staining for sGAG. Dark crimson colored regions shown in the middle of PGS and POC sections are scaffold materials. (A: 4× magnification, B: 10× magnification)

Figure 6

Immunohistochemical analysis for Type II collagen (brown) with hematoxylin staining (purple) (A: tissues between pores: 10× magnification, B: tissues inside a pore: 20× magnification, C: Outer layer tissues: 20× magnification).