The study results revolve around the long-established idea that machines within animal and human cells turn the sugars, fats, and proteins we eat into energy used by the body’s millions of cells.
The molecule most used to store that energy is called adenosine triphosphate, or ATP.
Along with this central role in metabolism, adenosine also helps signal other cells and serves as a building block of genetic material, and so is central to the growth of human tissue.
Previous research had shown that maintaining supplies of adenosine, known to nourish the chondrocyte cells that make cartilage, also prevented osteoarthritis in similar animal models of the disease.
In the new NYU Grossman School of Medicine-led study, researchers injected adenosine into the joints of rodents whose limbs had been damaged by inflammation resulting from either traumatic injury, such as a torn ligament, or from massive weight gain placing pressure on joints.
The biological damage in these cases is similar, researchers say, to that sustained in human osteoarthritis.
Publishing online in the journal Scientific Reports on Aug. 10, the study rodents received eight weekly injections of adenosine, which prompted regrowth rates of cartilage tissue between 50 percent and 35 percent as measured by standard laboratory scores.
“Our latest study shows that replenishing adenosine stores by injection works well as a treatment for osteoarthritis in animal models of the disease, and with no apparent side effects,” says lead study author Carmen Corciulo, Ph.D., a postdoctoral fellow at NYU Langone.
Corciulo says it is too soon to use this experimental model as a therapy in people.
Clinical trials must await a test drug that can be safely stored for days if not weeks, and experiments in larger mammals.
Study senior investigator Bruce Cronstein, MD, the Dr. Paul R. Esserman Professor of Medicine at NYU Langone Health, says the team’s research is important because the few existing drug therapies for osteoarthritis, such as acetaminophen and COX-2 inhibitor drugs, including naproxen and ibuprofen, only numb joint pain, or like hyaluronic acid, just lubricate its tissues.
None stall disease progression or reverse the damage. Painkillers, such as opioids, are often prescribed, but are also highly addictive, he cautions.
“People with osteoarthritis desperately need more treatment options with fewer side effects, and our research advances that effort,” says Cronstein, who also serves as the director of the Clinical and Translational Science Institute (CTSI).
He noted that other experimental medications are being developed elsewhere, including parathyroid hormone to stimulate bone growth, WNT inhibitor drugs to block the bone and cartilage degradation, and growth factor chemicals to promote cartilage growth.
Cronstein, Corciulo, and NYU Grossman School of Medicine have a patent application pending for the use of adenosine and other agents that help with its binding to chondrocytes, called A2A receptor agonists, for the treatment of osteoarthritis.
Among the study’s other key findings was that a cell-signaling pathway, known as transforming growth factor beta (TGF-beta) and involved in many forms of tissue growth, death and differentiation, was highly active in cartilage tissue damaged by osteoarthritis, as well as in cartilage tissue undergoing repair after being treated with adenosine.
Additional testing in lab-grown chondrocytes from people with osteoarthritis showed different chemical profiles of TGF-beta signaling during breakdown than during growth, providing the first evidence that the pathway switched function in the presence of adenosine (from assisting in cartilage breakdown to encouraging its repair.)
Developing treatments to halt or slow the disease is important, Cronstein says, because well over 100 million people worldwide are estimated to have osteoarthritis, which is tied to aging, especially in women.
This figure, he says, is only expected to grow as more people live longer and obesity rates climb.
“Right now, the only way to stop osteoarthritis is to have affected joints surgically replaced, which not only comes with pain and risk of infection, but is also quite costly,” says Cronstein.
“If new therapies can delay or prevent disease onset and progression, then fewer joint replacements will save people from a lot of pain and expense.”
The properties of articular cartilage allow for nearly frictionless motion in joints and the capacity to absorb large loads.1 Unfortunately, when cartilage is damaged, it cannot adequately repair itself to recover its prior function.1
Tissue engineering, a promising approach to repair damaged cartilage, currently falls short of creating functional tissue.2 Typically, tissue-engineered constructs exhibit inferior mechanical properties in comparison to native articular cartilage primarily due to the insufficient accumulation of extracellular matrix (ECM) components.3-6
Cartilage depends on mechanical forces for the development and maintenance of the ECM at a specific ratio of collagen to proteoglycans (typically between 2:1 and 3:1), which contributes to its unique mechanical properties.1,5
However, it is neither simple nor efficient to mechanically stimulate tissues in culture due to the limitations on the size and shape of constructs that can be grown.
Targeting the underlying mechanotransduction pathways responsible as a means to create functional tissue is an alternative strategy for tissue engineering that has been explored recently by our group.7
One molecule that appears to be integral to one particular chondrocyte mechanotransduction pathway is adenosine triphosphate (ATP), termed the purinergic receptor pathway.
ATP signaling has been implicated in mechanotransduction pathways in many different cell types including epithelial cells, astrocytes, osteoblasts, and chondrocytes.8
In articular chondrocytes, ATP is the first known molecule to be released as a result of mechanical stimulation and acts as an autocrine/paracrine signaling molecule.9
ATP acts on P2 receptors on the plasma membrane to promote ECM synthesis.10 While other nucleotides also have similar affinity for P2 receptors, such as uridine triphosphate (UTP) and ATP analogs,10 supplementation with exogenous ATP or UTP was shown to induce similar effects on ECM synthesis in bovine chondrocyte pellet cultures.11
Previously, we also observed that the addition of exogenous ATP to engineered cartilage cultures in vitro stimulated both collagen and proteoglycan synthesis and significantly improved the mechanical properties of the developed tissue.7
However, there appeared to be a dose effect of ATP on the cartilage cultures that warranted further investigation. High doses of ATP (250 µM), while eliciting increased matrix synthesis, did not result in increased ECM accumulation.7
Additionally, gene expression of matrix metalloproteinase 13 (MMP-13 or collagenase 3) was 2 times greater under high doses of ATP. Similarly, in other studies, high doses of exogenous ATP are known to evoke the release of inflammatory mediators,12,13 initiate matrix turnover,14,15 and induce mineralization.16
Undesirable effects, such as the mineralization of articular cartilage, have been associated with an accumulation of extracellular inorganic pyrophosphate (ePPi), which is a by-product of ATP degradation.17
In physiological media, low concentrations of ePPi acts to inhibit mineralization, while excess ePPi has been implicated in the formation of calcium pyrophosphate dihydrate (CPPD) crystals.17
Although the formation of CPPD crystals is poorly understood, these crystals can be a sign of pathological mineralization in the joint (chondrocalcinosis) and are associated with joint pain and subsequent cartilage degradation.18,19
There is evidence for 2 potential signaling pathways that connect the presence of CPPD crystals to increased matrix turnover. CPPD crystals either bind to toll-like receptors (TLRs) on the plasma membrane through a phosphatidylinositol 3kinase (PI3K)–dependent pathway20,21 or can be potentially endocytosed and elicit changes through a mitogen-activated protein kinase (MAPK)–dependent pathway.22
Therefore, the purpose of this study was to identify the mechanism of ATP-mediated catabolism and to determine a therapeutic ATP dose range for engineered cartilage to maximize the anabolic versus catabolic response.
Discussion
The application of mechanical loading to engineered cartilage constructs is a widely used method to enhance tissue growth and mechanical properties.3-6
Although this approach has been highly successful, there are limitations in applying mechanical stimuli to anatomically shaped constructs with irregular geometry and/or high radii of curvature.
Alternatively, by harnessing the known mechanotransduction pathways responsible, it may be possible to achieve the same effect in the absence of externally applied forces. In a recent study, we demonstrated that direct stimulation of the ATP-purinergic receptor pathway through exogenous supplementation of ATP can elicit a comparable anabolic response and be used to improve both tissue growth and mechanical properties of the developed tissue.7
However, high doses of ATP (250 µM) resulted in a simultaneous catabolic response characterized by an increase in MMP-13 expression, potentially due to the accumulation of ePPi.7
In the present study, we have determined a therapeutic dose range of exogenous ATP to maximize the anabolic response and ascertained that ATP-mediated matrix turnover was most likely a result of the formation and endocytosis of calcium-containing crystals from accumulated ePPi in the culture media.
ATP can be catabolized by soluble (e.g., tissue nonspecific alkaline phosphatase, tissue transglutaminase) and/or membrane-bound (e.g., nucleotide pyrophosphatase/ phosphodiesterases, ecto-5′-nucleotidase) nucleotide-degrading enzymes after P2 (purinergic) receptor binding, both leading to the formation of ePPi.
PPi, measured from the conditioned culture media after 4 weeks of nucleotide exposure, was significantly increased in response to ATP stimulation. This is consistent with previous studies that have also detected an increase in ePPi from conditioned culture media of both porcine explants and chondrocytes in monolayer culture as a result of P2 receptor signaling.30
There is a very narrow range for the physiological ePPi concentration in normal human articular cartilage, with synovial fluid in the knee containing 10 ± 0.5 µM PPi (determined over 50 individuals).31
The average basal level of PPi detected in the conditioned media surrounding 3-D cultures in the present investigation was similar to these values, at around 10 µM. Because 250 µM ATP supplementation increased MMP-13 activity along with the increased presence of ePPi, accumulated ePPi could have contributed to the catabolic effects observed under high doses of exogenous ATP.
PPi levels, determined from the digests of the 3-D cultured constructs, were also investigated to gain a better understanding of PPi homeostasis in the vicinity of cells.
No significant differences were observed in the amount of PPi present in the tissue digests, suggesting that excess ePPi was most likely being utilized and prompted our mechanistic investigation into how PPi was contributing to ATP-mediated catabolism.
In order to determine an optimal dose of exogenous ATP for the 3-D cultured constructs, additional doses flanking 62.5 µM were investigated (31.25 and 125 µM). MMP-13 activity was chosen as the measure of catabolism because it was the only matrix-degrading enzyme that was observed to be stimulated as a result of ATP stimulation.7
However, the limitation of this approach is that MMP-13 activity may not necessarily reflect all of the catabolic processes at work. Similarly, ECM synthesis determined at the end of the culture period (4 weeks) only reflects the anabolic response over the final 24-hour period after long-term ATP exposure.
For this reason, conclusions were derived by comparing patterns of MMP-13 activity and ECM synthesis with the long-term tissue formation and properties observed previously.7 The patterns for both ECM synthesis and MMP-13 activity as a result of ATP stimulation were strikingly similar.
ECM synthesis and MMP-13 activity were observed to increase with increasing ATP dose and stayed relatively stationary between 62.5 and 125 µM. Previously, it was observed that ATP-stimulated cultures (62.5 and 250 µM) yielded tissue with properties that were more indicative of native cartilage, with indentation moduli 5 to 6.5 times larger than the unstimulated controls.7
Although the long-term accumulation of collagen and proteoglycans was not investigated at 125 µM, the mechanical properties as a result of 62.5 and 250 µM did not significantly differ from each other, leading to the assumption that 125 µM ATP would elicit a similar effect on mechanical properties.
Therefore, the dose range of ATP between 62.5 and 125 µM was determined to be optimal for maximizing the anabolic effect and minimizing the catabolic effect of exogenous ATP for engineered cartilaginous constructs in our particular experiments.
It should be mentioned that due to interference with the MMP-13 activity assay, serum concentrations needed to be reduced in these studies. While this may affect comparison of the results, serum concentrations were only reduced during the final media change (48 hours) of the 4-week culture period, suggesting that any potential effects would be minimal.
Excess ePPi has been shown to induce an increase in MMP-13 gene expression in monolayer culture of bovine articular chondrocytes.20 In the present study, MMP-13 activity was increased in response to 10 µM PPi, but not 1 µM PPi, through a MAPK-dependent pathway.
This provides evidence that relatively small changes in ePPi concentration can lead to significant increases in MMP-13 activity of chondrocytes. Although PPi may appear to be a potential candidate for eliciting catabolism, its method of action is unclear.
There is no known direct route of entry for ePPi into the cell,17 and the accumulation of intracellular PPi as a result of multiple intracellular reactions32 is only cleared from the cytosol into the extracellular space by the 1-way transmembrane channel, progressive ankylosis protein (ANK).17,33
In addition, there is currently no known channel or receptor that actively transports or binds ePPi to induce intracellular signaling pathways.
When monolayer cultures were exposed to ePPi in calcium-free media, the previously observed PPi-mediated increases in MMP-13 activity did not manifest. This result implicates calcium and pyrophosphate as key factors involved in the PPi-mediated catabolic response. PPi mineralization could potentially explain this phenomenon.
CPPD crystals have been shown to spontaneously form at relatively low concentrations of PPi (>10 µM) in the presence of calcium (as well as being influenced by pH and the concentrations of sodium and magnesium).34 CPPD crystals can bind to TLRs, specifically TLR2, on the plasma membrane and elicit MMP-13 gene expression through a PI3K-dependent pathway20,21 or, alternatively, potentially be endocytosed and elicit changes through a MAPK-dependent pathway.22 Activation of the TLR pathway has been associated with inflammation, characterized by increased nitric oxide (NO) and prostaglandin E2 (PGE2) release.21
In our previous study, PGE2 and NO release was unaffected by ATP stimulation under all doses investigated (up to 250 µM).7 Similarly, inhibition of the PI3K-dependent pathway by LY294002 did not abolish the relative induction of MMP-13 activity from exogenous PPi.
Alternatively, inhibition of the MAPK-dependent pathway (specifically MEK1/2) by U0126 abrogated PPi-induced MMP-13 activity, suggesting that crystal endocytosis and activation of MEK1/2 (upstream of MAPK) could be the predominant mechanism of action of catabolism as a result of ATP stimulation.
A potentially confounding result was the lack of direct identification of CPPD crystals in the 3-D cultured constructs by TEM. However, it may be possible that CPPD crystals were first endocytosed and subsequently degraded, which has been demonstrated for hydroxyapatite crystal–induced metalloproteinase activity.35
The detection of CPPD crystals is also often fraught with difficulty18,36 because the crystals can be small and sparse, requiring more sophisticated methods of analysis.36 Amorphous forms of calcium pyrophosphate (more than 30 types have been identified) induced by excess PPi could also potentially elicit catabolic responses from chondrocytes through a similar mechanism.37
Studies that have positively identified CPPD crystals in articular cartilage as a result of exogenous ATP utilized significantly greater concentrations than what was used in the present study (>1 mM ATP)16,38,39 and specifically set out to induce a state of pathological mineralization or chondrocalcinosis (pathological presence of CPPD crystals in the joint space).19
Future work is required to confirm the presence of CPPD crystals in the 3-D cultured constructs. In addition, it should be noted that LY294002, in the absence of exogenous PPi, appeared to upregulate MMP-13 activity.
While the exact reason for this is currently unknown, PI3K signaling pathway is a major regulator of cell function and remodeling of the ECM and can influence other downstream signaling pathways involved in inflammation and matrix turnover.40
While future work is required to gain a better understanding of the effects of LY294002, inhibition of the PI3K signaling pathway had no apparent effect on the induction of MMP-13 activity in response to exogenous PPi.
These findings suggest that calcium-containing crystals, potentially formed from excess ePPi in the presence of Ca2+, may upregulate MMP-13 through a mechanism involving crystal endocytosis, and possibly subsequent dissolution, and the MAPK pathway.
If indeed CPPD crystals are causing increased MMP-13 activity as a result of ATP stimulation, mineralization inhibitors, for example, phosphocitrate,41 could potentially be utilized in conjunction with ATP to further extend the therapeutic range.
Phosphocitrate is a naturally occurring inhibitor of mineralization that has been used in several in vitro studies to inhibit both CPPD and hydroxyapatite crystal formation.41
Phosphocitrate acts by restricting the nucleation, growth, and aggregation of calcium salts41 and has also been shown to block crystal-induced MMP synthesis.42
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