Cyclic tensile strain enhances human mesenchymal stem cell Smad 2/3 activation and tenogenic differentiation in anisotropic collagen-glycosaminoglycan scaffolds

Abstract

Orthopaedic injuries, particularly those involving ligaments and tendons, are some of the most commonly treated ailments in the United States and are associated with both high costs and poor outcomes. Regenerative medicine strategies for tendon injuries could be enhanced by three-dimensional biomaterials that can promote cell alignment and pro-tenogenic differentiation of patientderived MSCs. We have previously described a collagenglycosaminoglycan (CG) scaffold possessing aligned structural features able to promote bone marrow MSC differentiation towards a tenogenic lineage, in the absence of growth factor supplementation. We aimed to employ a bioreactor to enhance MSC tenogenic differentiation within the aligned CG scaffold via cyclic tensile strain (CTS), and further to evaluate the relative effects of strain cycle duration and extended application of repeated cycles of CTS on MSC response. Human MSCs were cultured in CG scaffolds for up to 6 d under static (unloaded) or cyclic tensile strain (1 Hz) for 10 min every 6 h. Time-dependent activation of ERK 1/2 and p38 mechanotransduction pathways was observed within each 6 h strain cycle. MSCs remained viable throughout the experiment and application of CTS robustly upregulated the expression of tendon-specific extracellular matrix proteins and phenotypic markers. Simultaneously, CTS promoted increased phosphorylation of Smad 2/3, suggesting a link between tensile stimulation and TGF-β family growth factor production. Together, we demonstrated the design, fabrication and validation of a high-throughput tensile stimulation bioreactor to increase MSC tenogenic differentiation in porous CG scaffolds.

Decellularized extracellular matrix gelloids support mesenchymal stem cell growth and function in vitro

“…Briefly, 25 mg of native bovine muscle and solubilized D‐ECM were digested to elute double‐stranded DNA using a DNeasy assay kit (Qiagen). The eluent was used to quantify DNA content using a PicoGreen assay (Invitrogen) as previously described by Gilbert et al (Grier, Moy, & Harley, ).…”

Mineralized collagen scaffolds fabricated with amniotic membrane matrix increase osteogenesis under inflammatory conditions

“…Three of each cell-seeded mineralized collagen and mineralized collagen-amnion scaffold were lysed for protein at days 0, 4, 7, 14, 28. To extract protein, a solution of phosphate inhibitor cocktails (Sigma-Aldrich) and a RIPA lysis buffer were used (45). To evaluate the protein concentration, a Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific) and a Pierce TM Bovine Serum Albumin Standard Pre-Diluted Set (Thermo Fisher Scientific) were used.…”

“…If the power was below 0.8 for any sample set, then the data was deemed inconclusive. The number of samples used for each group was based on previous studies utilizing similar collagen scaffolds (44,45). compressive testing (n=8), pore size (n=3), metabolic activity (n=6), cell number (n=5), western blot (n=3), gene expression (n=5), and micro-CT and histology (n=3).…”

Anisotropic mineralized collagen scaffolds accelerate osteogenic response in a glycosaminoglycan-dependent fashion

“…26 We have previously developed an approach to fabricate non-mineralized collagen scaffolds with anisotropic pores to increase the metabolic activity of tenocytes and provide directional growth of these cells for tendon repair. [27][28][29][30][31] Further, glycosaminoglycans (GAGs) in the extracellular matrix play an important role in bone homeostasis and potentially regeneration. 32 GAGs linked to bone repair include chondroitin-6-sulfate (CS6), chondroitin-4-sulfate (CS4), and heparin sulfate (Heparin).…”