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

Abstract

Biomolecular environments encountered in vivo are complex and dynamic, with combinations of biomolecules presented in both freely diffusible (liquid-phase) and sequestered (bound to the extracellular matrix) states. Strategies for integrating multiple biomolecular signals into a biomimetic scaffold provide a platform to simultaneously control multiple cell activities, such as motility, proliferation, phenotype, and regenerative potential. Here we describe an investigation elucidating the influence of the dose and mode of presentation (soluble, sequestered) of five biomolecules (stromal cell-derived factor 1a , platelet-derived growth factor BB [PDGF-BB], insulin-like growth factor 1 [IGF-1], basic fibroblast growth factor [bFGF], and growth/ differentiation factor 5 [GDF-5]) on the recruitment, proliferation, collagen synthesis, and genomic stability of equine tenocytes within an anisotropic collagen-GAG scaffold for tendon regeneration applications. Critically, we found that single factors led to a dose-dependent trade-off between driving tenocyte proliferation (PDGF-BB, IGF-1) versus maintenance of a tenocyte phenotype (GDF-5, bFGF). We identified supplementation schemes using factor pairs (IGF-1, GDF-5) to rescue the tenocyte phenotype and gene expression profiles while simultaneously driving proliferation. These results suggest coincident application of multi-biomolecule cocktails has a significant value in regenerative medicine applications where control of cell proliferation and phenotype are required. Finally, we demonstrated an immobilization strategy that allows efficient sequestration of bioactive levels of these factors within the scaffold network. We showed sequestration can lead to a greater sustained bioactivity than soluble supplementation, making this approach particularly amenable to in vivo translation where diffusive loss is a concern and continuous biomolecule supplementation is not feasible.

Lactoferrin and parathyroid hormone are not harmful to primary tenocytes in vitro, but PDGF may be

“…When studied in small animal models of tendon healing, however, results were varied [29][30][31] . In this study, treatment of tenocytes with PDGF reduced the expression of the major tenocyte genes, scleraxis and tenomodullin, and previous studies also demonstrated that PDGF may have a detrimental effect on tendon gene expression 19 . Clinically, PDGF receptor expression is increased in diseased tendons, and is associated with hypercellularity 32 , and taken into context with our findings on the poor effects of PDGF on tendon-related genes, this suggests that PDGF may not be the most suitable factor for improving tendon healing outcomes.…”

“…The use of growth factors for treating tendon disorders has failed to have a clinical impact. PDGF is one of the most widely studied factors for improving tendon healing, and in vitro results have been especially positive, with potent increases in tendon cell and explant proliferation and collagen production observed 19,20,26 . When studied in small animal models of tendon healing, however, results were varied [29][30][31] .…”

“…Clinically, PDGF receptor expression is increased in diseased tendons, and is associated with hypercellularity 32 , and taken into context with our findings on the poor effects of PDGF on tendon-related genes, this suggests that PDGF may not be the most suitable factor for improving tendon healing outcomes. IGF-1 and TGF-β are both present within the tendon matrix 18 , and in vitro have been shown to be involved in tendon cell growth, collagen production and matrix remodelling [19][20][21][22][23][24][25] . Developmental studies suggest a key role for TGFβ1 in tendon development 33 , and inhibiting TGFβ1 has generally resulted in poor healing outcomes in in vivo tendon defect models 34 .…”

“…These were chosen as PDGF is among the most widely studied tendon factors, and IGF-1 and TGF-β are both present within the tendon matrix. Furthermore, all are upregulated during tendon healing, and are known to enhance tendon cell growth, collagen production and matrix remodelling, in vitro [18][19][20][21][22][23][24][25][26] . Furthermore, we have focussed on not only classical tendon-related outputs, such as cell proliferation and collagen production, we have also assessed the tenocyte gene expression profile, assessing genes important in tenocyte, chondrocyte and osteoblast biology.…”

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].…”