Methods of an Early Osteoporosis Diagnosis

 As of 2014, fifty-four million Americans had reported low bone density. Of these fifty-four million people, approximately ten million of them had been diagnosed with osteoporosis. Osteoporosis is a disease which simply means ‘porous bone’ and occurs when a person’s bones significantly lose mass and become porous, leaving them extremely fragile. This bone fragility increases the risk of bone breakages or fractures in the affected person. There are currently no real symptoms of osteoporosis; it is because of this, that the affected person is rarely diagnosed before the first fracture occurs. Though, researchers have been looking into potential biomarkers to assist doctors in the diagnosis of osteoporosis before the first fracture even happens.

The cell and membrane structure and function of cells involved in osteoporosis. The two cells which are responsible for bone formation and resorption are osteoblasts and osteoclasts, eukaryotic cells with multiple nuclei as well as a prominent mitochondria and Golgi apparatus (Caetano-Lopes et al., 2007). The membranes of both cells contain proteins such as –ATPase, IGF2R, TRAP, and cathespin K which are significant in the overall function of the cells (Ha et al., 2008). Osteoblasts, the cells which lay down new bone mineral do so by secreting a dense layer of collagen called the organic matrix. This secretion of matrix involves the deposition of an extremely dense hydroxyapatite-based mineral into the organic matrix to provide strength. This process of matrix secretion is driven by active and passive transport as well as pH control (Blair et al., 2017). Osteoclasts on the contrary, are the cells which break down the bone that is formed by osteoblasts. This resorption is promoted initially by endocytosis (ingestion of matter by the cell), followed by transcytosis (transportation of small molecules from one side of a cell to the other), of degraded bone matrix. This cycle of bone formation and resorption is called bone remodeling (Blair, 1998). These two very similar yet strikingly differently cells and their processes go hand-in-hand and when functioning properly, bone remodeling results in healthy strong bones.

The metabolic pathways, anabolic and catabolic, present within osteoblasts and osteoclasts. Mature osteoclasts are able to generate high ATP production rates due to their high mitochondrial and citric acid cycle respiration. This ATP is used during bone resorption. Researchers have found that glucose metabolism is actually increased while osteoclasts are differentiating. During this increased glucose metabolism, there is a shift towards mitochondrial respiration. This shift is what allows high ATP production, enhancing differentiation (Kim et al., 2007). For osteoblasts, studies show that the production of lactate from glucose without oxygen (aerobic glycolysis) is the main way for these cells to metabolize glucose. They also show that the lactic acid that is produced by glucose is stimulated by the calciotropic hormone parathyroid hormone (PTH).

This information has led most researchers to believe that the increase in lactic acid production is responsible for the increased active bone resorption (Esen et al., 2014). Osteoblasts contain Teriparatide, an available form of PTH, which increases bone turnover and the bone density of people affected by osteoporosis. The parathyroid hormone down regulates the expression of sclerostin, a protein encoded by the gene SOST, expression in osteocytes (osteoblast imbedded within the bone matrix) permitting the anabolic signaling pathway to proceed. The process ultimately improves skeletal microarchitecture by strengthening it (Silva et at., 2015). At the same time, the PTH receptor signaling in osteoblasts is responsible for increasing bone the ratio of RANKL/OPG (OPG is secreted by osteoblasts and prevents too much break down of bone matrix by osteoclasts through the binding of OPG with RANKL), which then in turn increases the recruitment of both osteoblasts and osteoclasts as well as osteoclast activity (resorption) (Boyce & Xing, 2007) (Nissenson, 2002). An example of these pathways being blocked in nature is the overexpression and/or inhibition of the Wnt signaling pathways (pathway made of proteins which pass signals through a cell). When this happens, the cell doesn’t know when to start or stop secreting bone matrix. If this happens, the tight roles of bone remodeling would not be balanced, increasing the risk of diseases such as osteoporosis (Rhee et al., 2013).

Cellular respiration and fermentation within osteoblasts and osteoclasts. As stated in previously, both osteoclasts and osteoblasts both require a great deal of ATP due to the fact that bone remodeling (bone resorption and formation) is a process which requires a lot of energy. However, not very much information on the processes these cells go through to meet this demand for such an extensive amount of energy is known as of now (Esen & Long, 2014) (Indo et al., 2013). Though the information on the process these two cells go through to generate ATP is limited, it is possible to piece together the data that has been collected by multiple researchers to piece together the possible processes of cellular respiration of two cells. Researchers have found that throughout the course of osteoclast differentiation, osteoclasts are capable of increasing the amount of ATP they produce in order to meet the large demand required for the bone resorption process. This is thought to be because during osteoclast differentiation, mitochondrial biogenesis, an increase in the mass of each cell’s mitochondria, is initiated in an attempt to increase ATP production as a response to the plethora of energy the cell is expending. Mitochondrial biogenesis is possible through the uptake of the iron transferrin receptor as well as the action of beta 1, a peroxisome proliferator-activated receptor-gamma coactivator. The combination of these two processes take part in the initial activation of cellular respiration through the provision of iron to the cells various respiratory proteins (Indo et al., 2013). We can assume the same goes to osteoblasts due to the fact that the two cells functions go hand-in-hand and are thought to have similar workings. Osteoblasts perform aerobic glycolysis with a goal of creating lactate. Though no articles were able to distinguish which pathway is used in the process, the production of lactic acid in osteoblasts is possible anaerobically (without oxygen) as well as aerobically (with oxygen).

Depending on the pathway being used, it is possible that osteoblasts perform lactic acid fermentation as well as aerobic glycolysis. Either way, the process of lactate formation is thought to play a key role in the overall function of the cell. This process of aerobic glycolysis is initiated when the Wnt signaling pathway and parathyroid hormone (PTH) activate osteoblast differentiation (Esen & Long, 2013). Similar to the fact that little information is known for sure about the processes osteoblasts go through to produce large amounts of ATP, little is known about the source of the high citrate levels in bones. It is thought by researchers that the high citrate levels may be caused by an altered metabolic reaction in the Krebs Cycle of cells known to produce greater net citrate (osteoblasts), that blocks the oxidation of citrate in the cell itself (Costello et al., 2012). This is just a theory at the moment, though it might be the cause of high citrate levels within bone tissue. As the osteoblasts mature, they begin oxidative phosphorylation in order to go through differentiation. The arrival of glucose and the process of oxidative phosphorylation allows an up-regulation in the amount of energy teach cell uses. This is necessary for the synthesis of collagen which is what allows differentiation to actually take place. After oxidative phosphorylation, the cell is capable of regulating the uptake of this glucose that is now available, through GLUT transporters (Gentur et al., 2014).

The different parts of cell communication within osteoblast and osteoclast cells. Once gain, based on the idea that osteoblasts and osteoclasts go hand-in-hand and work off each other and have very similar cellular makeups, it is possible to gather the information about the receptor proteins involved in one cell and ion gated channels involved in another, to piece together what the inner workings of both cells are like. Based on the results of an experiment they conducted, a group of researchers feel confident that the bone remodeling process and the presence of GPR103, a G-protein-coupled receptor, in cells involved in the bone remodeling are directly related. In this experiment, mice that were GRP103 deficient had a lower amount of osteoclasts compared to mice which contained a respectable amount of GRP103 G-protein-coupled receptors Baribault et al., 2006). If this proves to be true with time, organisms that are GRP103 deficient would possibly be less prone to osteoporosis due to the fact that their osteoblasts would be able to produce bone matrix at a higher rate than osteoclasts could break it down. Similar to this theory linking osteoclasts and GRP103, a theory about ligand gated ion channels present in osteoclasts was just discovered in 2001. It was found that the ligand gated channels present in osteoclasts consist of glutamate-gated channels as well as P2X nucleotide receptors.

These receptors are thought to act as targets for therapeutic agents with the purpose of treating bone disorders such as osteoporosis (Komarova et al., 2001). As or osteoblast cells, it was found that receptor tyrosine kinases and phosphorylation cascades are both involved in signaling within the cell. During the transition from pluripotent stem cells to osteoblasts, the expression of receptor tyrosine kinase Ror2 increases. Receptor tyrosine kinase Ror2 is responsible for inducing an osteogenic transcription factor, osterix, as well as well as promote the mineralization of bone matrix. Ror2 is also responsible for playing a major role in osteoblast differentiation (Liu et al., 2007). There is a possibility that by promoting mineralization and differentiation, the amount of mature osteoblasts increases. If this is the case, an increase in mineralization and differentiation can lead to a decrease in bone disease such as osteoporosis by creating bone matrix at a faster rate than bone resorption. The phosphorylation cascade involved in signaling in osteoblasts is when the MAPK pathway which is stimulated through the induction of MEK(SP), a constantly active form of MEK1, into MC3T3-E1 pre-osteoblast cells begin to differentiate and become osteoblasts. When MEK(DN), a dominant negative mutant, is introduced to the cascade, the process was inhibited and osteoblast differentiation did not take place (Xiao et al., 2000). This inhibition of the phosphorylation cascade could be a potential cause of osteoporosis due to the fact that osteoblasts are unable to differentiate and produce bone matrix. Along with the involvement of phosphorylation cascades and ligand gated ion channels in the signaling in osteoblasts, the cells use PTH to stimulate the secretion of neutral collagenase, an enzyme which plays a part in the the turnover of bone matrix as well as stimulate the signal transduction which involves protein-kinase C (PKC), making calcium ions second messengers used for signaling in osteoblasts (Civitelli et al., 1989).

The cell cycle of osteoblast cells. While osteoblasts and osteoclasts are very similar in that they are both somatic cells with 46 chromosomes per cell, the way they are formed is quite different. An osteoblast cell is formed from an osteogenic cell. Osteogenic cells are stem cells which are found within the tissue on the outer layer of bones, after these cells go through differentiation, they become osteoblasts. These cells reproduce asexually through mitosis and similar to most other cells spend a majority of mitosis in interphase in the G1 phase (Reece et al., 2014). It is during this period of time that cell cycle misregulation may take place. An example of cell cycle misregulation in ostegenic cells is the inhibition of proteasomes by MG132 blocks the cell cycle. By blocking the cell cycle, the down regulation of Runx2 in osteoblasts is no longer possible. In a study using mice, researchers concluded that mice which lack the protein Runx2 lack a mineralized skeleton. If this is the case in humans as well, the inhibition of proteasomes in osteogenic cells may cause a weaker and more fragile porous bone tissue (osteoporosis). It isn’t until the osteogenic cell completes mitosis and replicates that the cell goes through osteoblast differentiation and becomes an osteoblast (San Martin I et al., 2009).

The molecular basis of inheritance of osteoblast cells. Before the osteogenic cell splits to produce a second osteogenic cell, its DNA must first be replicated. The DNA replication process happens in osteoclasts the same way it would in any other cell. The double helix unzips with the help of helicase, gets RNA primers with the help of primase, and gets replicated by DNA polymerase, and the two strains of DNA are joined by ligase. If there are any mutations on the genes encoding the DNA replication machinery if osteoblasts such as the lack of the gene Recql4 during differentiation, it is likely that Rothmund Thomson Syndrome will become present in the person with this mutation. Rothmund Thomson Syndrome is linked with decreased bone mass in organisms. In a study which involved mice, it was found that mice which lacked the Recql4 gene had significantly lower bone mass than mice in which the Recql4 gene was present (Ng et al., 2015). Low bone mass causes weak porous bone, hence a lack of the Recql4 gene in humans is likely to cause osteoporosis.

The most interesting point of view in relation to Osteoporosis, osteoblasts, and osteoclasts. The point of view I found to be the most interesting for my topic was point of view 3. Point of view 3 was about cellular respiration and fermentation within osteoblasts and osteoclasts. The reason I chose this point of view as the most interesting is because based on the research I did on my two cells for this POV, there was very little definite answers to questions about the processes they go through and how they are able to do the things they do (Indo et al., 2013) (Esen & Long, 2014). The fact that researchers aren’t certain of how the bone matrix that is secreted from osteoblasts has such high citrate levels, is simply amazing to me (Costello et al., 2012).

What else I would like to know, and why researching osteoporosis from the point of view of the processes involved in cellular respiration is important. If I were to pursue this work further, I would want to know the answer to a question Dr. Kelly had brought to my attention while editing this POV, when osteoblasts perform aerobic and anaerobic glycolysis and produce lactic acid, what pathway is being used when the cell produced the acid anaerobically? Is this considered fermentation? When I looked into finding the answer to this question I didn’t come up with much, so it would be interesting to find the answer (Esen & Long, 2013).

I am also curious to know the exact process osteoblasts go through to produce bone matrix with such high levels of citric acid (Costello et al., 2012). I believe it is important for continued research to be done on osteoporosis from the point of view of cellular respiration and fermentation because this is the POV I found had the most unanswered questions and/or theories being looked into rather than known knowledge and I feel that if more research is done and we know more about the process osteoblasts and osteoclasts go through in order to do the things they do, we can use this information to try and find a way to better prevent or even cure bone diseases such as osteoporosis.