The influence of collagen–glycosaminoglycan scaffold relative density and microstructural anisotropy on tenocyte bioactivity and transcriptomic stability

Abstract

Biomaterials for orthopedic tissue engineering must balance mechanical and bioactivity concerns. This work describes the fabrication of a homologous series of anisotropic collagen–GAG (CG) scaffolds with aligned tracks of ellipsoidal pores but increasing relative densities (ρ*/ρs), and we report the role scaffold relative density plays in directing tenocyte bioactivity. Scaffold permeability and mechanical properties, both in tension and compression, were significantly influenced by relative density in a manner predicted by cellular solids models. Equine tenocytes showed greater levels of attachment, metabolic activity, soluble collagen synthesis, and alignment as well as less cell-mediated scaffold contraction in anisotropic CG scaffolds of increasing relative density. Notably, the lowest density scaffolds experienced significant cell-mediated contraction with associated decreases in tenocyte number as well as loss of microstructural integrity, aligned contact guidance cues, and preferential tenocyte orientation over a 14 day culture period. Gene expression analyses suggested tenocyte de-differentiation in the lowest density scaffold while indicating that the highest density scaffold supported significant increases in COMP (4-fold), tenascin-C (3-fold), and scleraxis (15-fold) expression as well as significant decreases in MMP-1 (9-fold) and MMP-13 (13-fold) expression on day 14. These results suggest that anisotropic scaffold relative density can help to modulate the maintenance of a more tendon-like microenvironment and aid long-term tenocyte transcriptomic stability. Overall, this work demonstrates that relative density is a critical scaffold parameter, not only for insuring mechanical competence, but also for directing cell transcriptomic stability and behavior.

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

“…This variant was chosen for its ability to prevent significant tenocyte-mediated contraction, along with the resultant loss of tenocyte alignment and downregulation of tenocyte-specific gene profiles, during long-term culture. 47 However, as they are three times denser than previous scaffolds, it is critical to examine strategies to improve tenocyte recruitment as well as subsequent proliferation and metabolic activity and compare the effectiveness of diffusion-mediated biomolecule transport versus sequestered biomolecules for enhancing the tenocyte response.…”

“…21 The anisotropic scaffold variant used throughout these studies was previously shown to support a high tenocyte bioactivity, while resisting a tenocyte-mediated contraction. 21,47 Scaffold crosslinking Dry scaffolds were dehydrothermally crosslinked at 105°C for 24 h under vacuum ( < 25 torr) in a vacuum oven (Welch) following lyophilization. Chemical crosslinking of scaffolds was achieved using carbodiimide chemistry with a solution of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC; Sigma-Aldrich) and N-hydroxysulfosuccinimide (NHS; Sigma-Aldrich) at a molar ratio of 5:2:1 EDC:NHS:COOH 48,49 for 1 h at 37°C.…”

“…47 RNA was reverse transcribed to cDNA using a Bio-Rad S1000 thermal cycler. Real-time PCR reactions were performed in triplicate with 10 ng of cDNA per reaction in an Applied Biosystems 7900HT Fast Real-Time PCR System.…”

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].…”

Role of tissue engineering in tendon reconstructive surgery and regenerative medicine: Current concepts, approaches and concerns

“…Also, the absorption rate is fast so that few days after implantation, the architecture is completely depleted by the inflammatory cells and mediators 2,12 . Moreover, the orientation of their polymerized fibres is randomized and is not suitable for the purposes of tendon tissue engineering 2,50,56 . Porous scaffolds are used to produce both bi-and tridimensional scaffolds with different purposes (Fig.…”