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The growth plate is a sandwich- like that entrapped between the epiphyseal and the metaphyseal bone at the ends of the long bones. Each growth plate contains a multilayer structure which can be divided into characteristic horizontal zones: the resting zone is the smallest zone; it contains primarily of small monomorphic chondrocytes with large nuclei and a narrow rim of cytoplasm (1). The cells in this zone do not show the columnar arrangement seen in other zones, but instead they found in small groups or rows which are usually oriented perpendicularly to the line of the columns (2). This zone is characterised by a high extracellular matrix to cell volume ratio and chondrocytes that have low rates of proliferation, proteoglycan and collagen type IIB production (1). These cells appear to play an essential role in the orientation of the underlying columns of chondrocytes which leads to unidirectional bone growth (3). The proliferative zone is characterised by the presence of chondrocytes arranged in particular characteristics columns parallel to the longitudinal axis of the bone. The cells in each column are separated from each other by matrices with large quantity of type II collagen. They are characterised by flattened appearance and a high mitotic activity. Additionally, these cells produce large amount of type II collagen as well as type IX and type XI collagen (4). Proteoglycan and other noncollagenous proteins such as cartilage oligomeric protein are also important components of the extracellular matrix in this zone (5).
At certain point, the proliferative chondrocytes stop replication and differentiate into prehypertrophic chondrocytes, coinciding with an increase in cell volume; these cells located in the transition zone. They then further differentiate into hypertrophic chondrocytes with rounded appearance. This differentiation is accompanied with a large increase in cell volume of about 5-10 fold (6). In comparison to other zones, the hypertrophic zone is composed of a distinctive extracellular matrix composition. The hypertrophic chondrocytes synthesize and secrete type X collagen, a unique short chain collagen confined to the hypertrophic zone, whereas collagen synthesis of type II, IX, XI is stopped or reduced (7). Hypertrophic chondrocytes also synthesize alkaline phosphatase which plays a role in the widening of the growth plate by increasing phosphate ion required for calcification (1). The hypertrophic chondrocytes synthesize different factors such as vascular endothelium growth factor (VEGF) which has a central role in the attraction of the invading blood vessels and osteoblastic cells (6). Around 80 % of the longitudinal septa, a matrix separates the longitudinal columns of chondrocytes which contains collagen fibrils aligned parallel to the long axis of the bone (8), are rapidly resorbed behind the invading blood vessels. While the remaining amount of the septa act as scaffold for the deposition of bone matrix by osteoblasts. Finally, the hypertrophic chondrocytes undergo chondroptosis and resorbed (9).
Regulation of the growth plate
The dynamic processes of the growth plate chondrocytes proliferation and differentiation leading to a distinctive morphological structure of the growth plate are highly regulated by the interaction of multiple systemic hormones, growth and mechanical factors. These cause an alteration in gene expression of chondrocytes in the growth plate.
The growth hormone (GH) and insulin like growth factor-I (IGF-I) are important regulators of the longitudinal bone growth. GH exerts its effects by acting directly and indirectly through the production of IGF-I. Somatomedin-hypothesis postulated by Salomon and Daughaday (1957) showed that GH stimulates IGF-I production in the liver. The latter then in turn activates chondrocytes proliferation in the growth plate (10). However, this view was challenged by other studies including a study by Isaksson et al (1982) who showed that local GH injection can stimulate tibial bone growth significantly (11). Later, a study by Nilsson et al (1986) demonstrated that local GH injection stimulates chondrocytes to express IGF-I in rats (12). GH appears to act on chondrocytes present in the resting zone, leading to local IGF-I production which causes a clonal expansion of proliferating chondrocytes in autocrine/paracrine manner (13). Chondrocytes in human and rabbit growth plats were found to express growth hormone receptors, supporting the direct effect of GH on the growth plate (14).
Chondrocytes proliferation, on the other hand, appears to be balanced by negative regulation via the binding of fibroblast growth factor (FGF) to FGF 3 receptors. Mutations in the FGF 3 receptors which normally expressed in the cells of the resting zone lead to the most form of dwarfism, achondroplasia and milder hypochondroplasia. These mutations result in continuous activation of these receptors leading to a decrease in chondrocytes proliferation. Therefore, the activation of these receptors seems to prevent cells entering the proliferative phase, most likely through the up-regulation of cell cycle inhibitors (15).
The proliferation of the growth plate chondrocytes also appear to be regulated by the feedback mechanism between parathyroid hormone related protein (PTHrP) and Indian hedgehog (Ihh). This feedback loop has an important role in determination when the cells leave the proliferative zone and enter the transition towards the hypertrophic zone. PTHrP is produced by the chondrocytes in the periarticular perichondrium. PTH/PTHrP receptors, however, are expressed predominantly in the prehypertrophic cells and the lower proliferating zone cells. The binding of PTHrP to PTH/PTHrP inhibits further differentiation of these cells into hypertrophic chondrocytes (16). Binding to PTH/PTHrP receptors stimulates two types of multiple heterotrimeric G proteins, Gs and Gq. Activation of Gs results in subsequent elevation of intracellular cAMP which causes the activation of protein kinase A (PKA) (17). The activation of PKA results in a decrease in p57, a cyclin dependant kinase inhibitor (18). Consequently, decreased p57 enhances chondrocytes proliferation and reduced expression of Runx 2, which causes a delay in chondrocytes hypertrophy (18). Additionally, the activation of PKA increases the activity of Sox 9, resulting in the inhibition of hypertrophy (19). The activation of Gq, however, results in an increase in chondrocytes differentiation but higher concentration is needed than that for Gs (20).
When prehypertrophic chondrocytes become far away from the PTHrP-producing cells, they can not be stimulated by PTHrP. Therefore, the prehypertrophic and the hypertrophic cells stop proliferating and begin to produce Ihh. The Ihh act to stimulate the production of PTHrP at the end of the bones (21). Ihh stimulates TGF-beta synthesis via acting on perichondrial cells, which in turn increase PTHrP production by perichondrial and periarticular cells (22). Furthermore, it has been demonstrated that TGF- beta can inhibit hypertrophy by acting directly on chondrocytes (23). Overall, the interaction between Ihh and PTHrP appears to be an important pathway for regulating proliferation and differentiation of the growth plate chondrocytes.
Thyroid hormones are also important for normal bone growth. Hypothyroidism is characterised by decreased rate of bone formation, a reduction in growth plate thickness of long bone and inhibition of hypertrophy (24). Administration of thyroid hormone, T4, on chondrocytes culture induces the expression of type X collagen, alkaline phosphatase activity and chondrocytes hypertrophy, suggestive the effect of thyroid hormones on chondrocytes differentiation (25). Additionally, T4 seems to be capable of entirely reverting reduced width of the proliferating and the hypertrophic zone (26). In comparison, T3 appears to stimulate the entry of germinal zone cells to the proliferating zone as well as enhancing growth plate chondrocytes differentiation (27).
Thyroid hormone can indirectly regulate bone growth by the stimulation of GH secretion and interacting with GH-IGF-I pathway. T3 was demonstrated to enhance proliferation of embryonic chicken chondrocytes by promoting IGF-I mRNA production (28).
Estrogen also appears to have a central role in controlling the growth plate acceleration as well as fusion in females and males (29). Estrogen, 17β-estradiol, alone is capable of inducing maturation of prehypertrophic chondrocytes into hypertrophic chondrocytes (30). This is accompanied with the synthesis of type X collagen and an elevation in alkaline phosphatase activity (30). This growth -promoting effect of estrogen appears to be primarily mediated via the stimulation of growth hormone GH -IGF-I axis and in part to growth hormone independent mechanism (31). In human, although low estrogen level accelerates growth, high doses of this hormone can suppress growth. Growth plate fusion appears to be mediated by higher level of estrogen, indicating that estrogen can also act as inhibitor for longitudinal bone growth (32). As well as acting indirectly, estrogen appears to act directly on chondrocytes. This was demonstrated by the expression of ER alpha and ER beta in growth plate chondrocytes (33).
Glucocorticoids appear to be a negative regulator of chondrogenesis. Dexamethasone treatment causes an inhibition of chondrocytes proliferation and matrix synthesis (34). Moreover, Hypertrophic chondrocytes express GC receptors, indicating that Glucocorticoids can act directly on these cells (35).
PI3K pathway appears to be essential in hypertrophic process and therefore in the longitudinal bone growth. Inhibition of these pathway results in 55% decrease in longitudinal bone growth; hypertrophic zone seems to be the most affected zone by the inhibition of PI3K, which results in a 45% reduction in the length of the hypertrophic zone (36).
Mechanical factors including exercise, compression loading and distraction have been demonstrated to alter the longitudinal bone growth and cellular activity of the growth plate. Sustained compressive loading appears to affect both the proliferative and the hypertrophic zones. A study by stokes et al (2007) on vertebral and proximal tibial growth plates showed that sustained loading causes a reduction of the growth plates by up to 53%. It was found that the reduction in growth plates was associated by changes in the number of the proliferative chondrocytes and the height of the hypertrophic chondrocytes (37). Distraction, however, causes an increase in the height of the proliferative and hypertrophic zones (38). However, few findings are present considering the effect of exercises at the physiological level of the growth plate. A study by Niehoff et al (2004) in which three groups of rats undergo three different physical activity levels showed no significant differences in the length of the femoral bone in the three groups of rats (39). However, other studies showed that exercises could increase or even decrease the longitudinal bone growth (40 and 41 respectively).
The role of IGF-1 and insulin in the growth plate and their effects on ATDC5 cells differentiation
Despite being reputed as mitogenic, a study by Wang et al (1999) demonstrated that a deficiency of IGF-I causes a 35 % reduction in the length of the hypertrophic zone but little change was observed in the proliferative zone, suggestive a role for IGF-I in the regulation of hypertrophy but not in proliferation. This was associated with a decrease in glucose transporter synthesis and glycogen production. This reduction in the hypertrophic zone length positively correlates with the reduced rate of longitudinal bone growth in IGF -I null mice (42). A Study on metastrals also supports the role for IGF-I in the hypertrophy process. This study showed that IGF-I increases the length of the hypertrophic zone by 3-fold which was accompanied by an increase in bone growth (43). IGF-mRNA was primarily detected in the hypertrophic chondrocytes and only few cells in the proliferative and the resting zones expressed IGF-I (44). Besides inducing hypertrophy, IGF-I can stimulate stem cell differentiation (45).
Studying differentiation using ATDC5, as an accepted model of chondrocytes differentiation, showed that IGF-I at physiological concentration as low as 10nm is able to induce differentiation (46). However, a study on growth plate foetal cow revealed that IGF-I mRNA was 32 fold more abundant in the proliferative chondrocytes than in the hypertrophic zone (47). In another study, IGF-I mRNA has been found mainly in the proliferative chondrocytes of mice growth plates (48)
Overall, the importance of IGF-I in longitudinal bone growth could be demonstrated by the major consequences associated with the deletion of IGF-I gene. It has been shown that transgenic mice with homozygous defect of IGF-I gene suffered from
profound embryonic and postnatal growth retardation (49).
In comparison, the importance of insulin in bone growth could be demonstrated by the consequences that occur in insulin deficiency as exemplified by diabetes. Diabetes appears to be associated with a reduction in bone growth especially in children at their prepubertal and pubertal periods (50). A study on diabetic rats showed that the growth plate width and endochondral bone growth of the proximal tibia were significantly reduced in untreated diabetic rats (51).
The effect of insulin on ATDC5 was demonstrated by Shukunami et al (1996) who showed that undifferentiated ATDC5 cells express both IGF-I receptors mRNA as well as insulin receptors mRNA, suggesting that insulin may induce differentiation of ATDC5 cells via an insulin-specific signalling pathway (52). This observation was consistent with subsequent study which showed that 50 nM insulin can exert half of the effects observed at the maximally differentiating concentration of 1600 nM; the latter is supposed to act through IGF-IR (53). This indicates that insulin can also act directly through insulin receptors (53). Studying the effect of insulin on foetal rat metatarsal showed that insulin concentration as high as 1600 nM has a greater potency for increasing bone length. However, 50nM insulin has a dramatic effect on the length of the hypertrophic zone (53).
Hypertrophic chondrocyte volume increase and its importance in longitudinal bone growth
The growth plate contains chondrocytes that are embedded in characteristic extracellular matrix. These cells are smaller and flatter at the epiphyseal end and then become larger and rounder towards the metaphyseal end. Ordered differentiation of these cells into three characteristic zones; the resting, proliferative and hypertrophic zones.
The relative proportion of the height of each zone appears to differ from species to another. A study on the proximal tibia of rats showed that the height of the resting, the proliferative and the hypertrophic zone account for 6%, 35% and 59% of total growth plate height of rats aged 21 days (54). However, a study on newborn ulnar porcine revealed that average proportion of the corresponding zones appeared to be 70%, 17 % and 13% respectively (55). This may indicate that from species to species each zone of the growth plate contributes differently to longitudinal bone growth.
Therefore, longitudinal bone growth appears to produce from a complex interplay between proliferation, hypertrophy, extracellular matrix and controlled degradation (56). The main contributor, however, to longitudinal bone growth appears to be cell enlargement during hypertrophy, accounting for about 44-59 % (57). The rest of the longitudinal bone growth is maintained by extracellular matrix and proliferation (57). Additionally, a study by Breur et al (1991) showed that a positive liner relationship exists between the longitudinal bone growth and the final volume of the hypertrophic chondrocytes (58).
Alteration in hypertrophic chondrocytes activities appears to have a significant regulatory role during acceleration and deceleration of the longitudinal bone growth. Hypertrophy appears to be a more efficient mean for producing bone growth than cell proliferation. In 35 day old rats, a columnar volume increase of a bout 15610 um3, terminal chondrocyte volume (17400 um3) minus end-proliferating cell volume
(1790 um3), was achieved by a volume increase in a single hypertrophic chondrocyte. However, production of nine cells is required, if the same volume increase is achieved by cell proliferation (54).
Knowing the importance of hypertrophic chondrocytes volume increase in longitudinal bone growth raises an interest of how this process is achieved. It appears that volume increase occurs by hypertrophy and swelling. The formal indicates the accumulation of dry matter mainly proteins, whereas the latter is simply the accumulation of fluids (59). Several studies support the notion that cell volume increase is predominantly occurred by swelling. Brighton et al in two different studies which were carried out using stereological techniques on fixed tissues showed that the increase in cell size was associated with an increase in the number and size of the holes in the cytoplasm as well as fragmentation of the cells membranes (60, 61). These findings were supported by a study done by Buckwalter et al, using morphological analysis of electron micrographs on fixed tissues, who showed that the volume of organelles per hypertrophic chondrocyte increased by 126 % ; whereas that of the cytoplasm and nucleoplasm increased by approximately 800 % most likely by swelling, suggesting the significant contribution of swelling to volume increase (62). The increase in the amount of cytoplasmic organelles including Golgi apparatus, endoplasmic reticulum, and mitochondria was considered important only in the early stages of chondrocytes enlargement (63).
On the other hand, Bush et al showed that the osmotic sensitivity of chondrocytes in the proliferative zone of the growth plate was not statistically significant from that in the hypertrophic zone. This indicates that hypertrophy plays a greater role in cell volume increase especially at the early stages of differentiation but swelling may have the greatest effect on cell volume increase in the last stages of differentiation (64).
The intracellular accumulation of organic osmolytes appears to contribute to osmotic pressure but only by 6-7% of the total cellular swelling during chondrocytes hypertrophy and inorganic osmolytes contribute less to this process (65). However, the contribution of these inorganic osmolytes appears to be considerably underestimated since hypertrophy and swelling require intracellular accumulation of osmolytes. Besides, for hypertrophy and swelling to occur, plasma membrane transporters synthesis must be increased (59).
Plasma membrane transporters
Chondrocytes are embedded in the extracellular matrix of cartilage which is characterised by unusual and variable osmotic and ionic environment. So as to regulate their volume in such environment, chondrocytes would necessitate expressing membrane transporter proteins for example sodium-potassium -chloride co- transporter (NKCC). The entry of Na+ during hypertrophic stress has a central role in the regulatory volume increase which is mediated by NKCC and other membrane transporters. The activation of these membrane transporters ultimately lead to increase in cell salt and water content (66).
NKCC appears to play an important role in the regulation of cell volume in most cells including growth plate chondrocytes. A study by Hunziker and Schenk (1989) has shown that morphological inspection of chondrocytes in situ revealed the high degree of co-ordination between matrix remodelling and chondrocytes shape change. This suggests that chondrocytes would necessitate to produce membrane transporters to increase fluid and electrolytes transport into the cytoplasm so as to regulate cell volume and electrolytes balance against the high osmotic and swelling pressure of the matrix (67).Wang Y et al (2004) demonstrated that genes encoded for certain membrane transporters are highly expressed in the hypertrophic zone than the proliferative zone particularly Slc12a2, gene which encode the secretory Na+-K+-2Cl co-transporter besides other genes involve in protein transports (68). The latter includes system A for amino acid transport and glutamate transporter (69). This indicates that these membrane transporters may play a central role in cell volume increase (69). Potassium channels could also have a role in the hypertrophic process; an outward-directed potassium channels have been found in the hypertrophic chondrocytes of chick growth plate (70).
Confocal laser scanning microscopy
The aim of this project is to investigate the changes in cell volume that occur during differentiation of growth plate chondrocytes and the effect of insulin and IGF-I on cell volume increase associated with differentiation. Therefore, cell volume can be measured using confocal laser scanning microscopy (CLSM) in which calcein acetoxymethyl (AM) is used as a volume marker. Calcein AM is neutral ester that is able to cross the plasma membrane and enter the cytosol. Cytosolic esterases hydrolyse calcein AM to calcein free acids that are membrane -impermeable, hydrophilic and intensely fluorescent. Calcein shows a high degree of fluorescence self quenching which means that the fluorescence emitted from calcein decreases at its high concentration but increases at its low concentration. This allows the changes in cell volume to be quantified at a low rate of photobleaching (reviewed in 66). Besides, chondrocytes loaded with calcein exhibit a high homogenous signal which is obviously defined (71). While the fluorescent calcein is retained by the cells with intact membrane, calcein rapidly leaks from the dead and damaged cells with compromised membranes.
Using CLSM, images will be taken for the cells, and cell volumes will be measured. However, to report the cell volume accurately cell edge needs to be determined correctly. This can be done using threshold segmentation method for determining the boundary of the cells. To estimate the threshold level, fluorescent fluoresbrite later beads of known diameter of 10.16 +- 0.1 will be imaged under identical conditions used for chondrocytes. The baseline threshold of 40 % may be used as previously reported (71).
Why ATDC 5 cells
Primary growth plate chondrocytes are needed in large amount in this project; therefore bovine growth plate is required. However, there is difficulty in obtaining bovine growth plate during undertaking the project experiments. This is because cows are usually bred in summer time since farmers try to time the birthing of the calves for the spring so that the claves are born outside and both the cow and the calf can benefit from fresh air. Besides, slaughter of calves normally done during spring and summer. Furthermore, separation of different subpopulations present in growth plates especially the prehypertrophic chondrocytes is technically challenging and required sophisticated techniques. Additionally, multiple passages of growth plate chondrocytes leads to a rapid loss of the differentiated phenotypes of chondrocytes (72).
Due to these limitations of using growth plate chondrocytes, clonal cell line capable of chondrogenic differentiation in vitro is required. Embryonic carcinoma cells are stem cells of tetracarcinoma which exhibit significant similarities to the cells of the early embryo. These cells lose their malignancy as they differentiate. Therefore, tetracarcinoma cells are very useful in the studying of the process of the early mammalian development (52).
ATDC5 cells are a clonal cell line that derived from the multipotent of embryonal carcinoma AT805 cells which have the ability to grow without feeder cells. The presence of insulin in culture causes these cells to differentiate into chondrocytes and form cartilage nodules at a very high frequency. These cells proliferate with a short doubling time of 16 hours regardless of the presence of insulin (52).
Differentiated ATDC5 cells express PTH/PTHrP receptors on their surfaces. These receptors are active and capable of activating adenylate cyclase (52). These receptors are highly expressed in the prehypertrophic cells when the cells convert from the proliferative to the hypertrophic stage (72). This finding was consistent with a study by Lee et al who showed that the expression of PTH/PTHrP mRNA peaks during the transition from the proliferating into the hypertrophic zone in foetal rat developing bone (73). Additionally, the differentiation of ATDC5 cells into hypertrophic cells is accompanied with the expression of type X collagen mRNA and the attenuation of type II collagen mRNA synthesis (72). Similar findings were demonstrated in a study on embryonic chick vertebral cartilage where hypertrophic chondrocytes showed an increase in the expression type X collagen mRNA, whereas the production type II collagen reduced (74).
The cellular and the extracellular morphology of the differentiated ATDC5 cells also show marked similarities with the hypertrophic chondrocytes observed in vitro and in vivo including the production of matrix vesicles, collagen and ALPase, all of which are involved in the mineralization process (72).
However, with respect to their chondrogenic potential, growth behaviour, morphological appearance and gene expression, ATDC 5 cells appears to keep the characteristics of chondrogenic precursor cells (52). But, overall ATDC5 cells provide an excellent model to understand the molecular mechanisms of longitudinal bone growth (72).
The growth plate is a highly specialized skeletal structure which is important for longitudinal bone growth. Longitudinal bone growth is mainly achieved by the actions of chondrocytes present in different proliferation and differentiation stages. These stages are regulated through various hormones, paracrine and mechanical factors which ultimately lead to bone elongation. Approximately 60 % of bone growth is due to hypertrophic chondrocyte volume increase (57), whereas the remainder is due to matrix production and proliferation. Cell volume increase in hypertrophic zone occurs because of hypertrophy and swelling. These mechanisms require the presence of plasma membrane transporters such as NKCC and other protein transporters. CLSM is used in this project to measure cell volume associated with differentiation and to determine the effect of insulin and IGF-I on cell volume increase.