"Introduction: Growth factors (GFs) are a key component of tissue engineering, but their exogenous administration has proven costly and ineffective. Extracellular matrix-inspired biomaterial approaches have sought to sequester these molecules, regulating their activity and presentation to cell receptors.1 Our previous work has shown that molecularly imprinted nanoparticles (MINPs) can play this role in standard 2D and 3D cell cultures, combining high recognition specificity, stability, and cost-effectiveness.2 Taking this concept a step forward, here we tested MINPs against transforming growth factor (TGF)-β3, a regulator of tenogenesis, in hydrogel systems with bioinspired ordered microstructures. Our hypothesis is that combined control over biophysical and biochemical cues will synergistically contribute to more robust tenogenic commitment of stem cells.
Methodology: A TGF-β3 N-terminal epitope was used as template molecule for solid phase imprinting by polymerization of acryloyl-containing monomers. MINP affinity and selectivity were assessed by surface plasmon resonance (SPR), Western blot and dot blot. Aligned polycaprolactone meshes were first produced by electrospinning, followed by cryosectioning into 50-µm microfibers. To enable their remote orientation within hydrogels, superparamagnetic iron oxide nanoparticles were synthesized by thermal decomposition and incorporated in the electrospinning solution. Finally, tenogenic constructs were prepared by encapsulating human adipose tissue-derived stem cells (hASCs), along with microfibers and MINPs, in transglutaminase-crosslinked gelatin hydrogels. Microfibers were unidirectionally aligned by applying a uniform magnetic field during gelation.
Results: SPR demonstrated a remarkable affinity of MINPs for the template (KD = 18±13 nM), in the range of some monoclonal antibodies. In comparison, the interaction between TGF-β3 epitope and MINPs imprinted against biotin was negligible, demonstrating the impact of imprinting on the molecular recognition potential of nanoparticles. hASCs remained viable for at least 14 days in hydrogel systems, showing a preferential orientation along the microfiber alignment axis. Preliminary qPCR findings indicate a positive correlation between MINP concentration and tendon-associated gene expression markers (SCX, TNMD) in aligned constructs, which is not observed in gels with randomly oriented microfibers. Furthermore, osteogenesis-associated ALP expression was downregulated as MINP concentration increased, corroborating the hypothesis of phenotypic steering toward tenogenesis. Protein synthesis is currently being analyzed by immunoassays to further bolster these results.
Conclusions: Our findings demonstrate the potential of molecular imprinting as a cost-effective complementary strategy in tissue engineering approaches. The endogenous GF sequestering ability of MINPs allows an efficient replacement of expensive recombinant GFs. Moreover, we also show that its combination with microstructural cues synergistically drives stem cell differentiation toward tenogenesis in engineered constructs. Thus, this strategy not only holds potential to significantly improve tendon healing after injury, but its principles can also be applied to engineer different tissues.
1. Teixeira, S.P.B. et al. Adv. Funct. Mater. 30, 1909011 (2020).
2. Teixeira, S.P.B. et al. Adv. Funct. Mater. 31, 2003934 (2021).
Acknowledgements: EU HORIZON 2020 for projects ACHILLES (H2020-WIDESPREAD-05-2017-Twinning-810850) and MagTendon (ERC-2017-CoG-772817); FCT/MCTES for scholarships PD/BD/143039/2018 (S.P.B.T.) under PhD-PATH (PD/00169/2013) and PD/BD/129403/2017 (S.M.B.) under PhD-TERM&SC (PD/59/2013), for project SmarTendon (PTDC/NAN-MAT/30595/2017), and individual contracts 2020.03410.CEECIND (R.M.A.D.) and CEECIND/01375/2017 (M.G.F.); Xunta de Galicia for postdoctoral grant ED481B2019/025 (A.P.).
Authors declare no conflicts of interest."