Articular Joint-Simulating Mechanical Load Activates Endogenous TGF-β in a Highly Cellularized Bioadhesive Hydrogel for Cartilage Repair

Abstract

Background: The treatment of osteochondral defects (OCDs) constitutes a major problem for orthopaedic surgeons. The altered mechanics and the cell types, with associated soluble factors derived from the exposed subchondral bone, are likely responsible for the mechanically and structurally inferior articular cartilage subsequently obtained as a repair tissue. There is therefore an unmet clinical need for bioresponsive biomaterials that allow cell delivery, reduce cell infiltration from the bone marrow, and support chondrogenesis in the presence of joint mechanical loading. Purpose: To develop a cell-laden injectable biomaterial, with bioadhesive properties, low cell invasion, and good mechanoresilience, in which simulated joint loading could induce tissue maturation through the production and activation of transforming growth factor beta 1 (TGF-β1). Study Design: Controlled laboratory study. Methods: Human bone marrow–derived mesenchymal stromal/stem cells were encapsulated in tyramine-modified hyaluronic acid (HA-Tyr) hydrogels, with crosslinking initiated by the addition of horseradish peroxidase (HRP) and various concentrations of hydrogen peroxide (H2O2; 0.3-2 mM). Cytocompatibility and biomechanical and adhesive properties were analyzed by live/dead staining, rheology, and push-out test, respectively. For multiaxial loading, cell-laden hydrogels were subjected to 10% compression superimposed onto a 0.5-N preload and shear loading (±25°) at 1 Hz for 1 hour per day and 5 times a week for 4 weeks. TGF-β1 production and activation were measured by enzyme-linked immunosorbent assay (ELISA). Results: The viscoelastic properties of the cell-laden HA-Tyr hydrogels, as crosslinked with different ratios of HRP and H2O2, were demonstrated for a range of cell densities and HRP/H2O2 concentrations. In the absence of serum supplementation, cell invasion into HA-Tyr hydrogels was minimal to absent. The bonding strength of HA-Tyr to articular cartilage compared favorably with clinically used fibrin gel. Conclusion: HA-Tyr hydrogels can be mechanically conditioned to induce activation of endogenous TGF-b1 produced by the embedded cells. HA-Tyr hydrogels function as cell carriers supporting biomechanically induced production and activation of TGF-β1 and as bioadhesive materials with low cell invasion, suggesting that they hold promise as a novel biomaterial for OCD repair strategies. Clinical Relevance: Leveraging physiological joint mechanics to support chondrogenic graft maturation in an optimized mechanosensitive hydrogel in the absence of exogenous growth factors is of highest interest for OCD repair.

Printability and Shape Fidelity of Bioinks in 3D Bioprinting

“… 125 Interestingly, for the enzymatic gelation of HA-tyramine, it was observed that the cell-induced decrease in viscoelastic properties diminishes at lower temperature, and the extent of this effect appears to vary with the embedded cell type. 152 This suggests that uptake and neutralization of reactive species might depend on the metabolic activity of the cells. A further aspect is that certain cross-linking chemistries, such as phenol–phenol coupling, thiol–ene or aldehyde-based systems, such as hydrazine-forming inks, 153 , 154 involve functional groups which are naturally present on amino acids and thereby on the cell surface.…”

Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels

“…For instance, the extraction of high-quality RNA from complex matrixes can be particularly challenging [12,13], especially with increasing culture duration, during which time increasing amounts of extracellular matrix are produced. This is also the case of the hyaluronic acid (HA)-based biomaterials commonly used in tissue engineering studies relevant to the musculoskeletal field [14][15][16]. The three dimensionality of a biomaterial-based culture, together with the composition of HA itself, likely influence the expression of reference gene candidates over a prolonged culture period.…”

Mammalian Cell Viability Methods in 3D Scaffolds for Tissue Engineering

“…As the authors of this chapter are active in the field of musculoskeletal research, examples will be illustrated using connective tissues or cells isolated from bone and joints. These fields often focus on joint-derived tissues that cause clinical problems for healing such as (i) anterior cruciate ligament (ACL) [1][2][3], (ii) cartilage scaffold engineering [4][5][6][7], (iii) intervertebral disc (IVD) regeneration [8][9][10] and (iv) bone regeneration [11][12][13][14][15]. We demonstrate a subset of fluorescent staining and methods to stain cells from bone and jointderived tissues: that is, staining mesenchymal stromal cells, chondrocytes and IVD cells in native tissue or in 3D hydrogel-like scaffolds such as fibrin [16], polyethylene glycol (PEG) [17], and also cells on solid materials such as silk [18].…”