Posts Tagged ‘bone remodelling’

Author: Aviral Vatsa PhD, MBBS

Nitric oxide (NO) is a short-lived, highly reactive, free radical which is ubiquitously present in the human body. Physiologically, it is widely used as a second messenger both an inter-cellular and intra-cellular signaling molecule. NO is produced when L-arginine is converted to L-citruline in the presence of NO synthase (NOS) enzyme, molecular oxygen, NADPH, and other cofactors. Principally, three isoenzymes of NOS are present in the body to catalyse the production of NO in various anatomic locations and under various physiological conditions. Three distinct genes encode for the three types of NOS i.e. endothelial (eNOS or NOS-3), neuronal (nNOS or NOS-1), and inducible (iNOS or NOS-2) NOS. Neuronal NOS and endothelial NOS are calcium-dependent enzymes, whereas inducible NOS is a calcium-independent inducible enzyme, that is activated and upregulated by cytokines during inflammatory processes. The tissue-specificity indicated in the names is not absolute as these subtypes have been discovered in wider locations in the body.

In bone, NO plays a vital role in mechanosensation and mechanotransduction. Osteocytes are widely accepted as the ‘professional’ mechanosensors in bone. They sense external mechanical loads on bone and produce chemical signals such as NO and prostaglandins. NO in turn has been shown to modulate the activity of both bone forming osteoblasts and bone resorbing osteoclasts. NO is essential for load-induced bone formation in vivo. Studies using single gene deletions have shown that NO is an important cog in the wheel for bone metabolism and bone remodelling. Although eNOS isotype is widely implicated in NO production in bone, but recent studies indicate that iNOS isotype might also be involved in NO production in bone in response to mechanical loading. Targeted deletion of eNOS shows mild osteoporotic phenotype in mice and iNOS pathway has been implicated in L-1-induced osteoclastic bone resorption. Hence both NOS isoforms have important role in bone remodelling.

Challenges to study NO: NO is a small, short-lived signalling molecule. It has a half life of less than 5 sec, which makes its online detection very difficult. Predominantly, the more stable metabolites of NO such as nitrites and nitrates are detected by using techniques such as Greiss reagent. They are however lited by the sensitivity levels and their inability to detect actual levels of NO. However, fluorescent dyes such as DAR 4M and DAF dyes are potent tools to detect online NO production at single cell level. These dyes are membrane-permeable, hence are taken up readily by the cells. Once inside the cell they are metabolised and rendered membrane-impermeable. When cell produces NO these dyes trap NO and get converted into fluorescent product, which can then be detected by using fluorescence microscopy. Moreover, by using these techniques, quantitative analyses of NO production (not only its metabolites) is feasible in live, single cells.

Molecular methods to investigate mRNA or protein levels of NOS enzymes are also used to corroborate with the changes in NO production levels.



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Author: Aviral Vatsa, Ph.D., MBBS

Bone is a highly dynamic tissue that responds to changes in its external environment. Our bones adapt their mass and architecture according to the external mechanical loading conditions. Any long term alterations in loading conditions result in alteration of bone mass and architecture. This is highlighted in the following examples:

  1. Astronauts tend to lose their bone when they are in space. This is because the bones are not mechanically loaded externally due to absence of or reduction in gravitational force.
  2. Tennis players gain more mass in their playing forearm as compared to the non-playing forearm.

In both these examples bones tend to readjust their internal structural mass and alignment as per the external loads or their absence. How bones can achieve this? How bone forming and bone resorbing cells can be orchestrated to bring about this adaptation?

Bone cells

The questions mentioned above can be answered by knowing more about the cellular components of bone and their functions. Our bones primarily have four cell types: osteocytes, osteoblasts, osteoclasts and bone lining cells. Osteocytes are believed to be the ‘professional’ mechanosensors of bone i.e. they sense the external loads put on bone. Osteoblasts are the bone forming cells. Osteoclasts are the bone resorbing cells and as the name suggests, bone lining cells line the bone surfaces and play a role in regeneration of osteogenic cells. Osteocytes, following mechanical loading, secrete signalling molecules such as nitric oxide (besides others). These signalling molecules then modulate the activity of bone forming osteoblasts and/or bone resorbing osteoclasts. Thus osteocytes orchestrate this process wherein adequate bone mass and architecture is achieved in accordance with the external loading conditions.

Anatomically, the osteocytes reside with in the hard bony matrix. They are the majority cell types in bone and are ideally placed to sense the mechanical loads. Osteocytes have a cell body and from the cell body arise nearly fifty cell processes. Through these cell processes each osteocyte forms a network with the surrounding osteocytes. Through this network, following mechanical loading, osteocytes can stimulate the activity of osteoblasts and inhibit the activity of osteoclasts. This process of maintenance of bone mass and architecture is called bone remodelling. Bone remodelling occurs through out our life. It occurs in response to microfractures, which can appear in our bone without being noticed clinically. As long as our bone metabolism is physiologically normal these stimuli, such as microfractures, result in bone remodelling.

In diseases such as osteoporosis, the mechanism of bone remodelling is disrupted and there is more bone resorbtion than new bone formation thus leading to reduction in bone mass and alteration of bone architecture. Drug therapies for osteoporosis such as bisphosphonates, act by inhibiting the activity of osteoclasts thereby resulting in reduction in bone resorbtion and hence helping in maintenance of adequate bone mass and architecture. Newer therapies that target to modulate a part of bone remodelling are being investigated.

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