Crosslinker concentration controls TGFβ-3 release and annulus fibrosus cell apoptosis in genipin-crosslinked fibrin hydrogels

Abstract

Back pain is a leading cause of global disability associated with intervertebral disc (IVD) pathologies. Discectomy alleviates disabling pain caused by IVD herniation without repairing annulus fibrosus (AF) defects, which can cause accelerated degeneration and recurrent pain. Biological therapies show promise for IVD repair but developing high-modulus biomaterials capable of providing biomechanical stabilisation and delivering biologics remains an unmet challenge. The present study identified critical factors and developed an optimal formulation to enhance the delivery of AF cells and transforming growth factor beta-3 (TGFβ-3) in genipin-crosslinked fibrin (FibGen) hydrogels. Part 1 showed that AF cells encapsulated in TGFβ-3supplemented high-modulus FibGen synthesised little extracellular matrix (ECM) but could release TGFβ-3 at physiologically relevant levels. Part 2 showed that AF cells underwent apoptosis when encapsulated in FibGen, even after reducing fibrin concentration from 70 to 5 mg/mL. Mechanistic experiments, modifying genipin concentration and integrin binding site presence demonstrated that genipin crosslinking caused AF cell apoptosis by inhibiting cell-biomaterial binding. Adding integrin binding sites with fibronectin partially rescued apoptosis, indicating genipin also caused acute cytotoxicity. Part 3 showed that FibGen formulations with 1 mg/mL genipin had enhanced ECM synthesis when supplemented with fibronectin and TGFβ-3. In conclusion, FibGen could be used for delivering biologically active compounds and AF cells, provided that formulations supplied additional sites for cell-biomaterial binding and genipin concentrations were low. Results also highlighted a need for developing strategies that protect cells against acute crosslinker cytotoxicity to overcome challenges of engineering high-modulus cell carriers for musculoskeletal tissues that experience high mechanical demands.

Mammalian Cell Viability Methods in 3D Scaffolds for Tissue Engineering

“…In the case of Bombyx mori silk, for instance, it was found that staining with EthD-1 was not possible due to a very strong autofluorescence of the silk material (Figure 9A and B). Moreover, integration of genipin, which is a natural cross-linker to increase stiffness of hydrogels, has been proposed [66][67][68][69]. The increasing concentration of genipin leads to increasing autofluorescence, noticeable as 'noisy background' on the red fluorescent channel.…”

Balancing biological and biomechanical performance in intervertebral disc repair: a systematic review of injectable cell delivery biomaterials

“…Several mechanisms can explain this ‘seesaw’ phenomenon. First, crosslinking reagents that are somewhat benign at low concentrations, can become cytotoxic to cells at high concentrations ( Cao et al ., 2012 ; Panebianco et al ., 2020 ; Saito et al ., 2008 ); thus, the amount of crosslinker required for a biomaterial to achieve the modulus of a load-bearing tissue like the IVD could be too high to simultaneously achieve cytocompatibility, motivating need for innovation. Second, increasing the concentration of macromers and additional crosslinkers reduces biomaterial porosity ( Brown and Barker, 2014 ; Cruise et al ., 1998 ), which can limit cell functionality by inhibiting nutrient transport, preventing cell-biomaterial interactions and physically constraining cells ( Fan and Wang, 2017 ).…”

Recent advances of hydrogel‐based biomaterials for intervertebral disc tissue treatment: A literature review

“…In the gelation step, some photo‐cross‐linked hydrogel may cause damage to cells due to the ultraviolet, while some chemical crosslinkers are potentially toxic to the encapsulated cells (Lin et al., 2013). An example is that genipin, the crosslinker in the FibGen, may cause AF cells apoptosis by inhibiting integrin binding (Panebianco, DiStefano, Mui, Hom, & Iatridis, 2020). When cells are encapsulated, nutrient diffusion related to the pore size and osmolarity of hydrogels should be well designed in case to impede cell proliferation and matrix synthesis (Krouwels et al., 2018; P. Li et al., 2016; Nativel et al., 2018).…”

“…In the gelation step, some photo-crosslinked hydrogel may cause damage to cells due to the ultraviolet, while some chemical crosslinkers are potentially toxic to the encapsulated cells (Lin et al, 2013). An example is that genipin, the crosslinker in the FibGen, may cause AF cells apoptosis by inhibiting integrin binding (Panebianco, DiStefano, Mui, Hom, & Iatridis, 2020).…”