Pralatrexate Sustainably Released from Polypeptide Thermogel Is Effective for Chondrogenic Differentiation of Mesenchymal Stem Cells.

Folic acid was reported to significantly improve chondrogenic differentiation of mesenchymal stem cells. In a similar mechanism of action, we investigated clinically approved antifolates by the U.S. Food and Drug Administration as chondrogenic-promoting compounds for tonsil-derived mesenchymal stem cells. A poly(ethylene glycol)-poly(l-alanine) thermogelling system was used as a three-dimensional cell culture matrix, where stem cells and antifolates could be incorporated simultaneously during a heat-induced in situ sol-to-gel transition. The antifolates could be supplied over several days by the sustained release of the drug from the thermogel. Initially, seven antifolates were prescreened based on cell viability and expression of a typical chondrogenic biomarker of type II collagen (COL II) at the mRNA level. Then, dapsone, pralatrexate, and trimethoprim were selected as candidate compounds in the second round screening, and detailed studies were carried out on the mRNA and protein expression of various chondrogenic biomarkers including COL II, SRY box transcription factor 9, and aggrecan. Three-dimensional cultures of stem cells in the thermogel in the absence of a chondrogenic promoter compound and in the presence of kartogenin (KGN) were performed as a negative control and positive control, respectively. The chondrogenic biomarkers were significantly increased in the selected antifolate-incorporating systems compared to the negative control system, without an increase in type I collagen (an osteogenic biomarker) expression. Pralatrexate was the best compound for inducing chondrogenic differentiation of the stem cells, even better than the positive control (KGN). Nuclear translocation of the core-binding factor ß subunit (CBFß) and enhanced nuclear runt-related transcription factor 1 (RUNX1) by antifolate treatment suggested that the chondrogenesis-enhancing mechanism is mediated by CBFß and RUNX1. An in silico modeling study confirmed the mechanism by proving the high binding affinity of pralatrexate to a target protein of filamin A compared with other antifolate candidates. To conclude, pralatrexate was rediscovered as a lead compound, and the polypeptide thermogel incorporating pralatrexate and mesenchymal stem cells can be a very effective system in promoting chondrogenic differentiation of stem cells and might be used in injectable tissue engineering for cartilage repair.

Thermogelling Inclusion Complex System for Fine-Tuned Osteochondral Differentiation of Mesenchymal Stem Cells.

How to control osteochondral differentiation of mesenchymal stem cells at a proper stage is a key issue for articular cartilage regeneration. To solve this problem, injectable scaffolds with different chemical functional groups were designed by introducing one equivalent of α-cyclodextrin (α-CD) carboxylate and α-CD phosphate along poly(ethylene glycol)-poly(l-alanine) (PEG-L-PA) block copolymers. Dynamic light scattering, transmission electron microscopy images, and two-dimensional NMR spectra indicated that the PEG-L-PA block copolymers formed inclusion complexes with α-CD derivatives. Aqueous solutions of PEG-L-PA block copolymers (P), α-CD carboxylate/PEG-L-PA block copolymers (PCC), and α-CD phosphate/PEG-L-PA block copolymers (PCP) underwent sol-to-gel transition as the temperature increased. The storage moduli of P, PCC, and PCP gels ranged from 1000 to 1300 Pa at 37 °C. Tonsil-derived mesenchymal stem cells (TMSCs) were incorporated in situ in the gel during thermogelation of P, PCC, and PCP, which became the three-dimensional cell culture systems with different functional groups. After 21 days of incubation of TMSCs in the P, PCC, and PCP systems, the chondrogenic differentiation biomarker of type II collagen significantly increased in the P system, whereas the osteogenic biomarkers of osteocalcin and runt-related transcription factor 2 significantly increased in the PCP system. Both chondrogenic and osteogenic biomarkers were highly expressed in the PCC system. This study proved that thermogelling inclusion complex systems consisting of PEG-L-PA block copolymers and α-CD derivatives could be an excellent injectable matrix for fine-controlling osteochondral differentiation of mesenchymal stem cells.