Chondrocytes-bioreactors

Bioengineered articular cartilage synthesized a drug for itself in response to the load

Oleg Lischuk, N+1

American researchers have created bioengineered articular cartilage based on pig cells, which produces an anti-inflammatory drug in response to mechanical stress. In the future, this technology can help in the treatment of osteoarthritis. The development report is published in the journal Science Advances (Nims et al., A synthetic mechanogenetic gene circuit for autonomous drug delivery in engineered tissues).

Osteoarthritis is the most common joint lesion, it affects every 30th inhabitant of the Earth, and the incidence increases sharply in old age. It occurs due to the gradual wear of articular cartilage, which is accompanied by inflammatory reactions and leads to the destruction of cartilage and the underlying bone area. This is due to the fact that after the final formation of the skeleton at the age of 20-30 years, cartilage cells (chondrocytes) practically cease to divide and restore cartilage tissue.

Nevertheless, these cells retain the ability to respond to stress, which is critical during the growth of the organism, for which a number of mechanoreceptors are responsible – TRPV4, PIEZO1, PIEZO2, integrins and primary cilia. A similar ability is used to create mechanotherapeutic bioengineering structures. Until now, such technologies have implied exogenous administration of peptide preparations, ultrasound stimulation or implantation of synthetic polymers (microcapsules, hydrogels), which do not provide long-term maintenance of drug concentration and its control by biofeedback.

To eliminate these shortcomings, the staff of Washington University in St. Louis and Duke University in Durham, led by Farshid Guilak, decided to create artificial cartilage tissue that will "self-heal" in response to mechanical stress.

In the course of preliminary experiments, the researchers isolated pig chondrocytes and incubated them in an artificial framework of agarose hydrogel, obtaining artificial cartilage. Such a tissue construct reacted to mechanical stress with a 108% increase in activation of calcium-dependent intracellular signaling pathways. The introduction of a low-molecular-weight inhibitor of the vanilloid cation channel of the type 4 transient receptor potential (TRPV4 receptor) GSK205 into the nutrient medium reduced such a reaction by 47 percent. This showed that the cellular response to mechanical stress primarily depends on this type of receptors.

By studying the reaction of single chondrocytes to osmotic stress (by the action of different nutrient media) and mechanical stretching of the membrane (by drawing into a micropipette), the researchers were convinced that the TRPV4-mediated reaction is observed only at the first exposure. That is, to get an answer, it is necessary to apply mechanical force to an entire sample of cartilage tissue with an osmotically active matrix, but not to individual cells.

Then the intracellular molecular response to TRPV4 activation was analyzed using the Ingenuity Pathway Analysis application and made sure that it includes a number of anabolic and inflammatory signaling pathways. Of these, the most promising for the creation of therapeutic transgenes were the activation circuits of the nuclear factor NF-kB and the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), which was confirmed by quantitative polymerase chain reaction.

Using genetic elements regulating NF-kB and PTGS2, the researchers created transgenes expressing the anti–inflammatory drug anakinru, a protein antagonist of interleukin-1 receptors, in response to TRPV4 activation. This drug, administered intravenously, has proven itself well in the treatment of rheumatoid arthritis and has proved effective in preclinical models of osteoarthritis. Nevertheless, his clinical trials for this disease did not bring the desired results, which may indicate the need for long-term controlled administration of the drug directly into the cartilage tissue.

The obtained therapeutic transgenes were injected into pig chondrocytes using a lentiviral vector and these cells were used to create bioengineered cartilage. In the experiment, such a construct with activated mechanoreceptors TRPV4 effectively produced anakinra and was not destroyed during treatment with interleukin-1 for 72 hours. Natural cartilage under the same influence lost 45.6 percent of sulfated glycosaminoglycans (the main component of the matrix that ensures the integrity of cartilage and its resistance to "wear"), and, accordingly, partially destroyed.

The principle of operation of bioengineered cartilage. A drawing from the article by Nims et al.

"We can create cells that automatically produce analgesics, anti-inflammatory drugs and growth factors for cartilage regeneration. In our opinion, such a strategy can become the basis for programming cells that have a therapeutic effect in a variety of medical problems," concluded Gulak.

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