Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are taken into account when performing in vivo studies, in vivo studies are still largely performed under static conditions.
We and others have shown that mechanical load can greatly influence chondroprogenitor and chondrocyte phenotype, generally beneficially, with factors such as TGF and nitric oxide being implicated. Taken together there is an increased need to implement mechanical stimulation during in vitro studies and to further understand the relative role of the specific load applied. This requires an increasing complexity of in vitro culture models, with the ultimate aim to recreate the articulating joint as accurately as possible. Building on previous studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs (1, 2), we have been further increasing the complexity of our in vitro/ ex vivo models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition (3). In addition, combining osteochondral defect models where viable cartilage and bone tissue can provide soluble cross talk, and a potential cell source, with an implanted therapeutic provides a multicellular environment more representative of the in vivo situation.
We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. While being able to apply multiple stimuli, selecting the stimuli for the vast number possible is a major challenge. Utilization of a design of experiment (DoE) approach can allow for a rapid screening of multiple factors, and their interactions, allowing for a smaller number to be selected for longer term studies. Design of higher throughput, multiwell multiaxial load bioreactors further increases the possibility to test multiple conditions or materials simultaneously opening more opportunities for in vitro screening. In vitro models have the major advantage that human cells from skeletally mature adults can be used, thus providing more clinically relevant data.
Improved understanding of the underlying mechanism of mechanically induced chondrogenesis can also assist with designing better protocols and streamlining biomaterial testing. Latent TGFβ can be mechanically activated and investigating the ability of cell free materials to activate TGFβ under mechanical load provides an insight to their performance under kinematic loading conditions (4).
The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable development pathways.
1. Gardner OFW, Fahy N, Alini M, Stoddart MJ. Joint mimicking mechanical load activates TGFbeta1 in fibrin-poly(ester-urethane) scaffolds seeded with mesenchymal stem cells. Journal of tissue engineering and regenerative medicine. 2017;11(9):2663-6.
2. Li Z, Kupcsik L, Yao SJ, Alini M, Stoddart MJ. Mechanical Load Modulates Chondrogenesis of Human Mesenchymal Stem Cells through the TGF-beta Pathway. J Cell Mol Med. 2010;14(6A):1338-46.
3. Monaco G, El Haj AJ, Alini M, Stoddart MJ. Sodium hyaluronate supplemented culture medium combined with joint-simulating mechanical loading improves chondrogenic differentiation of human mesenchymal stem cells. Eur Cell Mater. 2021;41:616-32.
4. Behrendt P, Ladner Y, Stoddart MJ, Lippross S, Alini M, Eglin D, et al. Articular Joint-Simulating Mechanical Load Activates Endogenous TGF-beta in a Highly Cellularized Bioadhesive Hydrogel for Cartilage Repair. Am J Sports Med. 2020;48(1):210-21.
This work was funded by the Swiss National Science Foundation (31003A_179438) and the AO Foundation.