Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress.

Ataxia-telangiectasia mutated (ATM) is a serine-threonine kinase that is integral in the response to DNA double-stranded breaks (DSBs). Cells and tissues lacking ATM are prone to tumor development and enhanced tumor cell migration and invasion. Interestingly, ATM-deficient cells exhibit high levels of oxidative stress; however, the direct mechanism whereby ATM-associated oxidative stress may contribute to the cancer phenotype remains largely unexplored. Rac1, a member of the Rho family of GTPases, also plays an important regulatory role in cellular growth, motility, and cancer formation. Rac1 can be activated directly by reactive oxygen species (ROS), by a mechanism distinct from canonical guanine nucleotide exchange factor-driven activation. Here we show that loss of ATM kinase activity elevates intracellular ROS, leading to Rac1 activation. Rac1 activity drives cytoskeletal rearrangements resulting in increased cellular spreading and motility. Rac1 siRNA or treatment with the ROS scavenger N-Acetyl-L-cysteine restores wild-type migration. These studies demonstrate a novel mechanism whereby ATM activity and ROS generation regulates Rac1 to modulate pro-migratory cellular behavior.

Characterization of Collagen Type I and II Blended Hydrogels for Articular Cartilage Tissue Engineering.

Biomaterials that provide signals present in the native extracellular matrix have been proposed as scaffolds to support improved cartilage regeneration. This study harnesses the biological activity of collagen type II and the superior mechanical properties of collagen type I by characterizing gels made of collagen type I and II blends. The collagen blend hydrogels were able to incorporate both types of collagen and retained chondroitin sulfate and hyaluronic acid. Cryo-scanning electron microscopy images showed that the 3:1 ratio of collagen type I to type II gels had a lower void space percentage (36.4%) than the 1:1 gels (46.5%). The complex modulus was larger for the 3:1 gels (G* = 5.0 Pa) compared to the 1:1 gels (G* = 1.2 Pa). The 3:1 blend consistently formed gels with superior mechanical properties compared to the other blends and has the potential to be implemented as a scaffold for articular cartilage engineering.

Incorporation of a Collagen-Binding Chondroitin Sulfate Molecule to a Collagen Type I and II Blend Hydrogel for Cartilage Tissue Engineering.

Adding chondroitin sulfate (CS) to collagen scaffolds has been shown to improve the outcomes for articular cartilage tissue engineering. Instead of physical entrapment or chemical crosslinking of CS within a scaffold, this study investigated the use of CS with attached collagen-binding peptides (termed CS-SILY). This method better recapitulates the aspects of native cartilage while retaining CS within a collagen type I and II blend (Col I/II) hydrogel. CS retention, average fibril diameter, and mechanical properties were altered by varying the number of SILY peptides attached to the CS backbone. When mesenchymal stromal cells (MSCs) were encapsulated within the scaffolds, the addition of CS-SILY molecules resulted in higher sulfated glycosaminoglycan production, and these results suggest that CS-SILY promotes MSC differentiation into chondrocytes. Taken together, our study shows the promise of adding a CS-SILY molecule to a Col I/II hydrogel with encapsulated MSCs to promote cartilage repair.

Collagen Type I and II Blend Hydrogel with Autologous Mesenchymal Stem Cells as a Scaffold for Articular Cartilage Defect Repair.

Collagen type II is a promising material to repair cartilage defects since it is a major component of articular cartilage and plays a key role in chondrocyte function. This study investigated the chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs) embedded within a 3:1 collagen type I to II blend (Col I/II) hydrogel or an all collagen type I (Col I) hydrogel. Glycosaminoglycan (GAG) production in Col I/II hydrogels was statistically higher than that in Col I hydrogels or pellet culture, and these results suggested that adding collagen type II promoted GAG production. Col I/II hydrogels had statistically lower alkaline phosphatase (AP) activity than pellets cultured in a chondrogenic medium. The ability of MSCs encapsulated in Col I/II hydrogels to repair cartilage defects was investigated by creating two cartilage defects in the femurs of rabbits. After 13 weeks, histochemical staining suggested that Col I/II blend hydrogels provided favorable conditions for cartilage repair. Histological scoring revealed a statistically higher cartilage repair score for the Col I/II hydrogels compared to either the Col I hydrogels or empty defect controls. Results from this study suggest that there is clinical value in the cartilage repair capabilities of our Col I/II hydrogel with encapsulated MSCs.