Harnessing the purinergic receptor pathway to develop functional engineered cartilage constructs

Objective: Mechanical stimulation is a widely used method to enhance the formation and properties of tissue-engineered cartilage. While this approach can be highly successful, it may be more efficient and effective to harness the known underlying mechanotransduction pathways responsible. With this aim, the purpose of this study was to assess the effect of directly stimulating the purinergic receptor pathway through exogenous adenosine 5\u27-triphosphate (ATP) in absence of externally applied forces. Methods: Isolated bovine articular chondrocytes were seeded in high density, 3D culture and supplemented with varying doses of ATP for up to 4 weeks. The effects on biosynthesis, extracellular matrix accumulation and mechanical properties were then evaluated. Experiments were also conducted to assess whether exogenous ATP elicited any undesirable effects, such as: inflammatory mediator release, matrix turn-over and mineralization. Results: Supplementation with ATP had a profound effect on the growth and maturation of the developed tissue. Exogenous ATP (62.5-250. μM) increased biosynthesis by 80-120%, and when stimulated for a period of 4 weeks resulted in increased matrix accumulation (80% increase in collagen and 60% increase in proteoglycans) and improved mechanical properties (6.5-fold increase in indentation modulus). While exogenous ATP did not stimulate the release of inflammatory mediators or induce mineralization, high doses of ATP (250μM) elicited a 2-fold increase in matrix metalloproteinase-13 expression suggesting the emergence of a catabolic response. Conclusions: Harnessing the ATP-purinergic receptor pathway is a highly effective approach to improve tissue formation and impart functional mechanical properties. However, the dose of ATP needs to be controlled as not to elicit a catabolic response. © 2010 Osteoarthritis Research Society International

Harnessing the purinergic receptor pathway to develop functional engineered cartilage constructs

Objective: Mechanical stimulation is a widely used method to enhance the formation and properties of tissue-engineered cartilage. While this approach can be highly successful, it may be more efficient and effective to harness the known underlying mechanotransduction pathways responsible. With this aim, the purpose of this study was to assess the effect of directly stimulating the purinergic receptor pathway through exogenous adenosine 5\u27-triphosphate (ATP) in absence of externally applied forces. Methods: Isolated bovine articular chondrocytes were seeded in high density, 3D culture and supplemented with varying doses of ATP for up to 4 weeks. The effects on biosynthesis, extracellular matrix accumulation and mechanical properties were then evaluated. Experiments were also conducted to assess whether exogenous ATP elicited any undesirable effects, such as: inflammatory mediator release, matrix turn-over and mineralization. Results: Supplementation with ATP had a profound effect on the growth and maturation of the developed tissue. Exogenous ATP (62.5-250. μM) increased biosynthesis by 80-120%, and when stimulated for a period of 4 weeks resulted in increased matrix accumulation (80% increase in collagen and 60% increase in proteoglycans) and improved mechanical properties (6.5-fold increase in indentation modulus). While exogenous ATP did not stimulate the release of inflammatory mediators or induce mineralization, high doses of ATP (250μM) elicited a 2-fold increase in matrix metalloproteinase-13 expression suggesting the emergence of a catabolic response. Conclusions: Harnessing the ATP-purinergic receptor pathway is a highly effective approach to improve tissue formation and impart functional mechanical properties. However, the dose of ATP needs to be controlled as not to elicit a catabolic response. © 2010 Osteoarthritis Research Society International

ATP promotes extracellular matrix biosynthesis of intervertebral disc cells

A recent study by our lab found high accumulation of extracellular adenosine triphosphate (ATP) in the center of healthy porcine intervertebral discs (IVD). Since ATP is a powerful extracellular signaling molecule, extracellular ATP accumulation may regulate biological activities in the IVD. Therefore, the objective of this study was to investigate the effects of extracellular ATP on the extracellular matrix (ECM) biosynthesis of porcine IVD cells isolated from two distinct anatomical regions: annulus fibrosus (AF) and nucleus pulposus (NP). The ATP treatment significantly promoted the ECM deposition and corresponding gene expression (aggrecan and type II collagen) by both cell types in 3-dimensional agarose culture. A significant increase in ECM accumulation was found in AF cells at a lower ATP treatment level (20 µM) compared to NP cells (100 µM), indicating that AF cells may be more sensitive to extracellular ATP than NP cells. NP cells also exhibited higher ECM accumulation and intracellular ATP than AF cells under Control and treatment conditions, suggesting that NP cells are intrinsically more metabolically active. Moreover, the ATP treatment also augmented the intracellular ATP level in NP and AF cells. Our findings suggest that extracellular ATP not only promotes ECM biosynthesis via molecular pathway but also increases energy supply to fuel that process