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Importance of Osteoprotegerin in regulating bone density.Introduction:BackgroundBones are complex tissues in living organisms especially vertebrates. (9) The composition of the bone matrix can be categorized into two components. These are the organic components which cover up 40% of the dry weight along with inorganic components which cover up rest of the 60%. The organic components can be further subdivided into collagen, proteoglycans, matrix proteins and cytokines and growth factors. Collagen is the most abundant protein that contributes to 90% of the organic component. There are mainly twenty different types of collagen, however only collagen I, II and III are the ones that mainly contribute to more than three-fourths of the different types listed. Bones mainly carry type I collagens that are composed of one alpha-2 and two alpha-1 chains. These are responsible for providing tensile strength. Compressive strength is provided by proteoglycans which are made up of glycosaminoglycan-protein complexes. Matrix proteins, on the other hand, are mainly non-collagenous proteins that are used to increase the bone formation as well contribute to mineralization. Cytokines and growth factors are present in the matrix but small quantities. They mainly help in bone cell differentiation, growth, activation. IL-1, IL-6, IGF, and IGF-beta, BMPs are examples of cytokines and growth factors. The inorganic component includes osteocalcium phosphate and calcium hydroxyapatite which also helps to provide compressive strength. (7) (8). Bones play a vital role in carrying out the necessary functions in a living organism especially vertebrates as invertebrates mostly do not have a skeleton of bone. It is mainly responsible for providing mechanical support, and it also aids in the protection of the organs. It is one of the reasons why a living organism is capable of maneuvering from one place to another. These two being the main features of a bone, it is also a store of various minerals and calcium which is an important element as it helps the nerve, muscle, and cells function normally. It is because of the collaboration of these important body parts that provide for the health of bones. Apart from these certain functions, one other vital function is to act as a biological environment for hematopoiesis or the formation of blood cellular component. (9) According to (Nijweide et al.1986) the growth, development, and maintenance of bone, all these are considered as highly regulated processes. (10) The balance between bone formation and resorption plays a fundamental role in maintaining bone mass. Osteoblasts (bone forming cells) and Osteoclasts (bone resorption cells) are the key players in this highly regulated step (9) OsteoblastThese are single nucleus cells that collectively work in the process of bone formation. They originate from mesenchymal stem cells (11), and they are majorly involved in bone reshaping. Dense crosslinked collagen, specialized proteins in minute quantities along with osteocalcin and osteopontin are formed by osteoblasts. These cumulatively help in the composition of the organic matrix of the bone. OsteoclastsThese are defined as large multinucleated cells which are located in the pits of the bone surfaces. These bone surfaces are also known as reabsorption bays. The size of a human osteoclast is about 150-200 µm in diameter, and they consist of 5 nuclei. Large cells (100 µm) consisting of dozens of nuclei come into existence when osteoclast inducing cytokines are utilized in the conversion of macrophages into osteoclasts. Since these do not have a non-natural substrate, this specific type of cells is characterized differently from other living bone cells. (1,2) There is a very high concentration of vesicles and vacuoles in osteoclast. This is the reason why osteoclasts have a homogenous foamy like cytoplasm. These vacuoles consist of lysosomes which are filled with acid phosphatase which is an enzyme commonly present in many plant and animal species. (3) This allows the characterization of tartrate resistant acid phosphatase and cathepsin K. (4,5,6).The significant role of osteoclasts is to be involved in the breakdown of bone tissue which is vital due to maintenance, repair, and remodeling of bones of the vertebral skeleton. Problems related to osteoporosis and osteopetrosis.Large category of hormones, inflammatory mediators and growth factors are involved in the regulation of both osteoblasts and osteoclasts (12,13,14). If there is an imbalance in the functions of these two cells, it can cause skeletal abnormalities along with conditions such as osteopetrosis and osteoporosis. Osteopetrosis is the increase in bone mass, and osteoporosis is the decrease in bone mass. (9)Old people suffer from fractures due to Osteoporosis. It is also a reason why the trabecular bone is degraded more often than the cortical bone. (25) According to statistics, it has been seen that 1.3 million fractures occur in the United States of America and the prime reason for this is osteoporosis. 25% of the Women who are aged 65 or more are more likely to suffer from one or multiple vertebral fractures. There are some reasons which can lead to osteoporosis. One of the most leading causes can be a lack of calcium in the diet. Due to deficiency of calcium in the diet, calcium is withdrawn from the bones which are known to be the central store of calcium in the body. Although a certain threshold for the recommended calcium has not been listed, it is suggested that at least 800mg/day are required for adults and 1200mg/day are necessary for growing adolescents. A recent statistic has shown that women lack this certain threshold in their diet. Other causes of osteoporosis can be an excessive amount of phosphate in the diet. An increased protein intake and insufficient vitamin D intake can also lead to osteoporosis. (25)On the contrary, osteopetrosis also called marble bone disease is the opposite of osteoporosis. It is a rare inherited disorder. This causes the bones to become more dense and hard. Although this might seem like a positive impact on the anatomy of the human beings but in reality, it is far more fatal than it is assumed to be.  It can have severe consequences which mainly involve breakage or dissolving of the bones. (26)Osteoprotegerin and its significanceA study was conducted on mice regarding increased bone mass or osteopetrosis. (9,15). This study led to some strong advances regarding the regulation of bone mass. (9,15) Initiating from genetic abnormalities or defects in osteoclast development, maturation, and activation which leads to a decrease in bone resorption resulting in osteopetrosis to the essential or vital roles being played by the microenvironment in the differentiation of osteoclast. (9,16). All these factors along with signal transduction necessary for the bone resorption that is mediated by osteoclast to the gene expressions playing a significant role in osteoclast development. All these points were brought about in the study that was conducted on mice. (9,17,18,19). According to Rodan and Martin (1981), the maturation and activation of osteoclast are regulated by osteoblast-derived factors during remodeling. (9,20). This phenomenon proved that genes play a vital role in the regulation of bone mass. (9)According to the study conducted by W.S Simmon, a particular element involved in the regulation of bone mass was discovered. It was named osteoprotegerin (OPG). This means to protect the bone. Osteoprotegerin is a protein which belongs to the tumor necrosis factor receptor (TNFR) superfamily. This was first identified during a fetal rat intestine sequencing project by sequence homology. This particular family consists mainly of membrane proteins that evoke signal transduction in different kinds of cells. Osteoprotegerin ended up expressing osteopetrosis linked up with a decrease in osteoclast. This meant an augmentation of the bone mass combined with a decrease in bone resorption cells. (9)  When the sequencing project was carried out the cDNA had encoded 401 amino acid polypeptides. They had certain characteristics which were found to exist in glycoproteins. This meant having a peptide which would be hydrophobic. There were four potential sites of N-linked glycosylation. The N terminals of the proteins were very much similar to all the other members of the superfamily especially TNFR-2 and CD40. The only anomaly that osteoprotegerin had was that it did not have the hydrophobic transmembrane-spanning sequence. The C terminal of Osteoprotegerin did not show any similarities to other proteins or protein motifs. This data is proof that the OPG protein indeed is a member of the TNFR superfamily. Research has also shown that the mouse and human OPG proteins are 85% to 94% identical to the OPG proteins discovered in mice. (9)Northern Blotting was the technique used to identify 3.0kb sized OPG transcripts. They were found in the mice or rodent tissues especially in the liver, lungs, heart, and kidneys. OPG mRNA was also seen to be expressed in the stomach. (9) It was also found in the intestines along with the skin and calvaria. According to W.S Simmons, in human tissues, a transcript of similar size is detected at the highest levels in the lung, heart, kidney, and placenta. Detectable levels were also shown in various hematopoietic and immune organs. (9)According to research conducted by (Simonet et al., 1993), a human apolipoprotein E gene promoter was genetically introduced into mice. These transgenic mice were later on shown to express the OPG cDNA. There was no big difference detected between the animals expressing the OPG protein and the ones that weren’t. Radiographs of the animals had shown an increase in the radiodensity of the long bones along with various other parts of the skeletal systems. It is often seen that the introduction of a foreign gene can cause abnormalities in the transgenic animals. Although no abnormalities were detected in bones, splenomegaly was one such condition that had occurred in the transgenic animals. Splenomegaly is a phenomenon where the spleen becomes enlarged. (21,17,18)Recombinant OPG also helps to block osteoclastogenesis which is the formation of osteoclasts or bone resorption cells. In a study conducted, with the aid of an osteoclast forming assay, two different situations were analyzed. Both the transgenic mice along with its control was used to determine the outcome of the experiment. The first condition was the absence of exogenous OPG. This led to results which clearly depicted the spleen cells in both the transgenic mice and its control to contain osteoclast precursors. A follow-up experiment was conducted, but this time it was done in the presence of murine OPG at levels of 100 and 10ng/ml. Osteoclastogenesis was completely inhibited in both the transgenic mice and the control. It is seen that recombinant OPG also led to the increase of bone density in vivo as augmented levels of circulating OPG is considered to be the reason for an increase in the bone and cartilages. OPG was injected into non-transgenic mice on a daily routine. After seven days it was observed that there was an increase in the trabecular bone in the area of the tibial metaphysis. The trabecular bone which is also known as cancellous bone is a type of porous bone.Recombinant Osteoprotegerin also plays another role in protecting the skeletal structure of the bones in ovariectomized rats. An ovariectomized rat is a female rat which is considered to be the ideal choice when conducting research. It is strongly preferred over rabbits and sheep due it being cost-effective and convenient to handle. (22) The ovariectomized rats have its ovaries removed. One very significant problem mostly found in ovariectomized rats is that they can suffer from bone loss due to estrogen deficiency caused by ovariectomy. This particular condition in ovariectomized rats can be compared to the bone loss caused by estrogen deficiency in human adults. (22)  This problem is visible especially during the early stage of osteoporosis. (23). The stages are the increased rate of bone turnover, which leads to less bone formation followed by more resorption. The next stage is a rapid bone loss phase which is followed by a much slower phase. It also leads to higher loss of cancellous bone in comparison to that of the cortical bone. The cancellous bone which is also known as the spongy bone is softer, weaker and more flexible in comparison to cortical bone. The cortical bone is responsible for forming the extremely hard exterior of the bones. The surface area of the cancellous bone is larger when in comparison to the cortical bone. Another important step in the early stages of osteoporosis is the reduced intestinal calcium absorption. The research was conducted on fisher rats that were 12 weeks old. They were initially ovariectomized. After four days they were treated for two weeks with recombinant osteoprotegerin derived from rodents. The results turned out to be positive. Recombinant osteoprotegerindid help to protect the skeletal structures of the bones from bone loss caused by a decrease in estrogen.DiscussionWith all the functions of osteoprotegerin determined via the following experiments, it is safe to assume that osteoprotegerin(OPG) is strongly responsible for bone density regulation. It not only helps to inhibit osteoclast maturation but it also acts as a solution to problems like osteoclast remodeling along with bone loss due to ovariectomy. Its identity as one of the members of the superfamily TNFR has also been proved via the experiments conducted. Although osteoprotegerin has been known to be a very strong determinant in keeping the structures of the bones intact, elevated levels of this protein, however, has shown to act negatively on the coronary artery. (27) Apart from the bone, the osteoprotegerin is developed in the cardiovascular system which includes heart, arteries, veins. (9) A study was conducted on 201 patients. Many risk factors such as sex, hypertension, diabetes, hyperlipidemia and current smoking habits were not taken into consideration while determining whether elevated OPG levels were related to coronary artery diseases or not. It was found that the OPG levels were very high in patients with clinical stenosis of the coronary arteries. Stenosis, in the medical field, is another term to describe the narrowing down of a certain body channel. When the level of this is clinically high, it can pose a problem. Therefore, the conclusion was that increasing serum OPG levels did lead to coronary artery diseases. (27) With this, it can be concluded that although OPG can be advantageous in helping to keep the bone structure intact, it does have its drawbacks if the levels go beyond the mark as the body functions as a whole system which needs to be kept balanced from all angles. This proves that something that can be beneficial to one part of the body can be detrimental to the other if a balance is not involved.  

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