The effect of anisotropic collagen-GAG scaffolds and growth factor supplementation on tendon cell recruitment, alignment, and metabolic activity

Abstract

Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freezing temperature, we created a homologous series of aligned CG scaffolds with constant relative density and degree of anisotropy but a range of pore sizes (55–243 μm). Equine tendon cells showed greater levels of attachment, metabolic activity, and alignment as well as less cell-mediated scaffold contraction, when cultured in anisotropic scaffolds compared to an isotropic CG scaffold control. The anisotropic CG scaffolds also provided critical contact guidance cues for cell alignment. While tendon cells were randomly oriented in the isotropic control scaffold and the transverse (unaligned) plane of the anisotropic scaffolds, significant cell alignment was observed in the direction of the contact guidance cues in the longitudinal plane of the anisotropic scaffolds. Scaffold pore size was found to significantly influence tendon cell viability, proliferation, penetration into the scaffold, and metabolic activity in a manner predicted by cellular solids arguments. Finally, the addition of the growth factors PDGF-BB and IGF-1 to aligned CG scaffolds was found to enhance tendon cell motility, viability, and metabolic activity in dose-dependent manners. This work suggests a composite strategy for developing bioactive, 3D material systems for tendon tissue engineering.

The past, present and future in scaffold-based tendon treatments

“…Advancements in engineering, chemistry and biology have made available numerous technologies that allow fabrication of hierarchical threedimensional scaffolds that closely imitate native tendon architectural features and mechanical properties, whilst enabling localised and sustained delivery of therapeutics [350]. Collagen sponges, for example, with or without aligned tracks and loaded with GAGs, growth factors and various cell populations have demonstrated enhanced cell motility and phenotype maintenance in vitro and increased collagen expression levels in small animal models [351][352][353][354][355]. However, such scaffold conformations cannot provide adequate mechanical resistance, in such a high mechanical demand environment [356].…”

Composite Growth Factor Supplementation Strategies to Enhance Tenocyte Bioactivity in Aligned Collagen-GAG Scaffolds

“…9,21,46 Anisotropic CG scaffold fabrication via freeze-drying Scaffolds were fabricated via directional solidification as previously described. 21 Briefly, degassed CG suspension was added to a polytetrafluoroethylene (PTFE)-copper mold and placed on a precooled ( -10°C) freeze-dryer shelf (VirTis). The CG suspension was frozen at -10°C for 2 h, and then sublimated at 0°C and 200mTorr to remove ice crystals.…”

“…[13][14][15][16][17] CG scaffolds have also been adapted for a variety of orthopedic applications covering osteochondral, bone, and tendon tissue engineering. [18][19][20][21] We have recently demonstrated a directional freeze-drying method to create geometrically anisotropic CG scaffolds with aligned tracks of ellipsoidal pores that mimic elements of native tendon anisotropy. 21 While we showed that the platelet-derived growth factor BB (PDGF-BB) and the insulin-like growth factor 1 (IGF-1) promoted increased tenocyte proliferation and migration within these anisotropic scaffolds, 21 a major concern remains the trade-off between increased proliferation and retention of the tenocyte-associated phenotype.…”

“…[18][19][20][21] We have recently demonstrated a directional freeze-drying method to create geometrically anisotropic CG scaffolds with aligned tracks of ellipsoidal pores that mimic elements of native tendon anisotropy. 21 While we showed that the platelet-derived growth factor BB (PDGF-BB) and the insulin-like growth factor 1 (IGF-1) promoted increased tenocyte proliferation and migration within these anisotropic scaffolds, 21 a major concern remains the trade-off between increased proliferation and retention of the tenocyte-associated phenotype. 22 Soluble factor supplementation is a widely applied scaffold enhancement strategy and has shown considerable functional value for tendon tissue engineering.…”

“…23,24 PDGF-BB and IGF-1 have previously been shown to promote dosedependent increases in tenocyte proliferation and collagen synthesis, both individually and in concert. 21,22,[25][26][27] Additionally, both factors have been noted as chemoattractants of tenocytes 21 as well as mesenchymal stem cells (MSCs). [28][29][30] This study aimed to assess the individual and combined effects of a wider range of soluble factors on cell viability, protein synthesis, and gene expression in the context of tendon tissue engineering.…”