Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD)

Abstract

Osteoarthritis, a degenerative disease of the load-bearing joints, greatly reduces quality of life for millions of Americans and places a tremendous cost on the American healthcare system. Due to limitations of current treatments, tissue engineering of articular cartilage may provide a promising therapeutic option to treat cartilage defects. However, cartilage tissue engineering has yet to recapitulate the functional properties of native tissue. During normal joint loading, cartilage tissue experiences variations in osmolarity and subsequent changes in ionic concentrations. Motivated by these known variations in the cellular microenvironment, this study sought to improve the mechanical properties of neocartilage constructs via the application of hyperosmolarity and transient receptor potential vanilloid 4 (TRPV4) channel activator 4α-Phorbol 12,13-didecanoate (4αPDD). It was shown that 4αPDD elicited significant increases in compressive properties. Importantly, when combined, 4αPDD positively interacted with hyperosmolarity to modulate its effects on tensile stiffness and collagen content. Thus, this study supports 4αPDD-activated channel TRPV4 as a purported mechanosensor and osmosensor that can facilitate the cell and tissue level responses to improve the mechanical properties of engineered cartilage. To our knowledge, this study is the first to systematically evaluate the roles of hyperosmolarity and 4αPDD on the functional (i.e., mechanical and biochemical) properties of self-assembled neotissue. Future work may combine 4αPDD-induced channel activation with other chemical and mechanical stimuli to create robust neocartilages suitable for treatment of articular cartilage defects.

Transient Receptor Potential Vanilloid 4 as a Regulator of Induced Pluripotent Stem Cell Chondrogenesis

“…Increases in chondrogenic marker expression, matrix production, and cell proliferation were not observed in the GFP− (nonchondrogenic) cells after GSK101 stimulus, possibly due to the distinct Ca 2+ signaling response of these cells, or due to this heterogeneous cell population having already differentiated into other cell lineages. The enhanced biosynthesis of GFP+ cells is consistent with previous studies showing that stimulation of TRPV4 with mechanical, osmotic, or chemical activators results in a strong anabolic response in primary chondrocytes, characterized by significant increases in extracellular matrix synthesis and accumulation 22,38,39 . It is important to note that while mechanical factors can enhance chondrogenesis, 40 the ability to accelerate cartilage tissue formation using a small molecule chemical agonist may have significant advantages over the use of growth factors or direct mechanical stimulation (eg, in a bioreactor), particularly for large, inhomogeneous, or anatomically shaped tissue grafts 41,42 …”

“…Not only was Trpv4 expression and function associated with chondrogenesis, we also observed that activation of TRPV4 signaling in with mechanical, osmotic, or chemical activators results in a strong anabolic response in primary chondrocytes, characterized by significant increases in extracellular matrix synthesis and accumulation. 22,38,39 It is important to note that while mechanical factors can enhance chondrogenesis, 40 the ability to accelerate cartilage tissue formation using a small molecule chemical agonist may have significant advantages over the use of growth factors or direct mechanical stimulation (eg, in a bioreactor), particularly for large, inhomogeneous, or anatomically shaped tissue grafts. 41,42 The specific mechanisms involved in TRPV4 enhancement of chondrogenesis remain to be determined.…”

Tension stimulation drives tissue formation in scaffold-free systems

“…TRPV4 has previously been shown to be an important mechanosensitive ion channel that mediates the cellular response to compressive stimulation and ultimately affects neotissue functional properties 21 ; without a functioning TRPV4 channel, compressive stimulation did not increase the compressive modulus. In our hands, TRPV4 agonism resulted in up to a 153% increase in tensile properties 36 , 37 . Here we showed that TRPV4 channel function is needed to sense tension stimulation and initiate signal transduction to result in functional property increases (Young's modulus), as GSK205 inhibition of TRPV4 during tension stimulation eliminated the emergent tissue-level response to tension ( Figure 3b ).…”

Challenges in Fabrication of Tissue-Engineered Cartilage with Correct Cellular Colonization and Extracellular Matrix Assembly

“…Since there is no provided ECM to offer bulk to the construct, a high number of cells is usually required to produce appreciable tissues [ 114 , 116 , 117 ]. Similarly, as there is no ECM to guide the cells, there is often a need for mechanical and chemical stimulation of the cultures in order to guide ECM production [ 116 , 121 , 122 ]. This also means that the start of the culture can be precarious for the cell phenotype as there are no physical signals coming from the ECM.…”