In vitro generation of mechanically functional cartilage grafts based on adult human stem cells and 3D-woven poly(epsilon-caprolactone) scaffolds

Abstract

Three-dimensionally woven poly(epsilon-caprolactone) (PCL) scaffolds were combined with adult human mesenchymal stem cells (hMSC) to engineer mechanically functional cartilage constructs in vitro. The specific objectives were to: (i) produce PCL scaffolds with cartilage-like mechanical properties, (ii) demonstrate that hMSCs formed cartilage after 21 days of culture on PCL scaffolds, and (iii) study effects of scaffold structure (loosely vs. tightly woven), culture vessel (static dish vs. oscillating bioreactor), and medium composition (chondrogenic additives with or without serum). Aggregate moduli of 21-day constructs approached normal articular cartilage for tightly woven PCL cultured in bioreactors, were lower for tightly woven PCL cultured statically, and lowest for loosely woven PCL cultured statically (p<0.05). Construct DNA, total collagen, and glycosaminoglycans (GAG) increased in a manner dependent on time, culture vessel, and medium composition. Chondrogenesis was verified histologically by rounded cells within a hyaline-like matrix that immunostained for collagen type II but not type I. Bioreactors yielded constructs with higher collagen content (p<0.05) and more homogenous matrix than static controls. Chondrogenic additives yielded constructs with higher GAG (p<0.05) and earlier expression of collagen II mRNA if serum was not present in medium. These results show feasibility of functional cartilage tissue engineering from hMSC and 3D-woven PCL scaffolds.

Figure 1

The 3D-woven PCL scaffold. (A) schematic; (B–C) scanning electron micrographs of (B) loosely and (C) tightly woven scaffolds. Scale bars: 1 mm.

Figure 2

(A) Aggregate and (B) Young’s moduli of 21-day constructs based on hMSC and either loosely or tightly woven PCL scaffolds, and cultured either statically or in bioreactors. *Significant difference due to scaffold structure; **Significant difference due to culture vessel.

Figure 3

Amounts of (A) DNA, (B) total collagen, and (C) glycosaminoglycans (GAG) in constructs produced from tightly woven scaffolds and hMSC cultured for up to 21 days, statically or in bioreactors in DM1, statically in DM2, and statically in CM. ^aSignificant difference due to type of culture vessel, ^bSignificant difference due to serum, ^cSignificant difference due to chondrogenic additives.

Figure 4

Histological appearance of constructs produced from tightly woven scaffolds and hMSC cultured for 21 days statically in DM1 (column 1), in bioreactors in DM1 (column 2), statically in DM2 (column 3) or statically in CM (column 4). Rows I–II: full cross-section (I) or en-face (II–III) sections stained with safranin-O/fast green (GAG appears orange-red, cell nuclei black, and PCL scaffold white); Row IV: en-face sections double immunostained for collagen types I and II (Coll-II appears green, Coll-I was stained red and was not seen, DAPI-counterstained cell nuclei appear blue. Scale bars: 200 µm (Row I); 50 µm (Row II); 200 µm (Row III); 100 µm (Row IV).

Figure 5

Electrophoresis gels for RT-PCR products of collagen type II and of Sox-9. Lane 1= day 0 hMSCs; Lanes 2 and 3 = 7 days and 21 days static culture in DM1; Lanes 4 and 5 = 7 days and 21 days bioreactor culture in DM1; Lanes 6 and 7 = 7 days and 21 days static culture in DM2; Lane 8=control for DNA contamination.