Gel-like carrier materials support in vitro chondrogenesis of mesenchymal stem cells and improve ectopic stability of stem cell-derived cartilage in vivo
Andrea Dickhut, Eric Steck, Wiltrud Richter
Orthopaedic University Hospital Heidelberg, Heidelberg, Germany
[email protected]
Introduction: Gel-like carrier materials were introduced into cell therapy of
cartilage lesions to improve chondrocyte retention and distribution in the defect.
Mesenchymal stem cells (MSC) are now discussed as an alternative cell source for
repair but their differentiation to chondrocytes may be hampered by carrier materials. We here asked whether distinct gel-like carriers can support chondrogenesis
of MSC in vitro and lead to stable cartilage-like transplants in vivo.
Materials and Methods: MSC derived from human adipose tissue were
embedded in collagen type I gel, fibrin glue, Matrigel and PuraMatrix peptide
hydrogel and cultured under chondrogenic conditions with TGF-beta 3 and BMP6 for up to 6 weeks. For histological evaluation, differentiated constructs were
embedded in paraffin, and sections were analyzed by alcian blue staining and collagen type II and X immunohistology. Cell viability was investigated by staining
with FDA/PI. Expression of Col2A1, Col10A1 and alkaline phosphatase mRNA
was evaluated using quantitative RT-PCR. Proteoglycan content was measured by
extraction of alcian blue dye, and incorporation of 3H-proline was used as a measure for total collagen synthesis. Alkaline phosphatase activity of cell culture supernatants was determined by hydrolysis of p-nitrophenylphosphate. After 5 weeks of
chondrogenic induction, differentiated constructs were transplanted subcutaneously into SCID mice. Four weeks later explants were analyzed by histology for collagen type II, proteoglycans, and calcification by immunohistology, alcian blue staining, and alizarin red staining respectively.
Results: All carriers supported cell viability and chondrogenesis in vitro.
Quantitative RT-PCR revealed material-dependent differences in Col2A1 and
alkaline phosphatase mRNA expression, but not in Col10A1 expression. The carrier materials displayed differential effects on total collagen synthesis and proteoglycan deposition and enhanced in vitro differentiation under suboptimal conditions with strongest enhancement observed for PuraMatrix. Alkaline phosphatase
activity measured in cell culture supernatants was reduced by all carrier-materials
compared to carrier-free pellet cultures. Four weeks after transplantation, ectopic
transplants were positive for collagen type II and proteoglycans (Fig. 1 A-F). The
undesired calcification seen in carrier-free ectopic transplants (Fig. 1 G) in vivo was
also present in collagen and fibrin constructs, while Matrigel consistently suppressed calcification of ectopic transplants (Fig. 1 H). For PuraMatrix constructs a
calcification was found for 2 of 4 donor cell populations (Fig. 1I).
Figure 1. Collagen type II (A, B, C), alcian blue (D, E, F) and alizarin red (G, H, I) staining
of paraffin sections of MSC constructs 4 weeks after transplantation into SCID mice. Before
transplantation, constructs were cultured for 5 weeks under chondrogenic conditions with 10
ng / ml TGF-beta 3 and 10 ng / ml BMP-6. (A, D, G) Carrier-free pellet; (B, E, H) Matrigel;
(C, F, I) PuraMatrix. Transplants were positive for collagen type II (A, B, C) and rich in proteoglycans (D, E, F) but calcification was found in carrier-free pellets (G) as well as in collagen and fibrin (not shown). In PuraMatrix constructs calcification was only observed for two
out of four donor cell populations (I) while all Matrigel constructs showed no calcification (H).
Discussion: Our study indicates that gel-like biomaterials are suitable carriers
for MSC in cartilage repair. All materials tested in this study supported MSC differentiation into chondrocytes, but displayed differential effects on distinct parameters of chondrogenesis and ectopic in vivo stability. Promotion of chondrogenesis
at suboptimal conditions and suppression of alkaline phosphatase activity and calcification makes particular gel-like carrier materials even more attractive for MSCbased tissue engineering approaches for regeneration of cartilage lesions.
Poster No. 570 • 54th Annual Meeting of the Orthopaedic Research Society