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Endochondral Ossification Essay

A) Growth plate and Endochondral ossification: In order to maintain a healthy skeletal system which is crucial for different organs such as respiratory system, brain, spinal cord and heart, it is very important to have a good development of the skeletal system. Endochondral ossification is the process through which the axial and appendicular skeletons are formed through the formation of a cartilage inter-mediate. A good model for endochondral ossification is the limb bud where the precursor cells of the mesenchyme condense and then followed by the differentiation of the most central chondrocytes.

Endochondral ossification is a highly regulated process by different signalling like the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with important roles in development and disease. (1) Endochondral ossification is an organized process that starts during fetal life, and continues until growth stops in early adulthood. Different cell lines are involved in the endochondral ossification process yet, it is the chondrocyte that drives the process (2).

There are several paracrine factors that regulate the different processes of the differentiation of chondrocytes, their proliferation, the secretion of cartilage matrix, cell death, and of vascular and bone cell invasion . Among these factors are the insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), Indian hedgehog (IHH) and parathyroid hormone-related protein (PTHrP), bone morphogenic proteins (BMPs), WNTs, and vascular endothelial growth factors (VEGFs). Moreover there is a series of endocrine factors that control the rate of endochondral bone formation at the growth.

These factors include growth hormone (GH), IGF-I, thyroid hormone, glucocorticoids, androgens, and estrogens (3). Talking about the physiology of the growth plate, it resembles a sandwich-that is formed of multilayer structure that is divided into three layers: the resting zone, proliferative zone and the hypertrophic zone (4) fig 1. Different species have different relative proportions of the three zones (5). Figure 1:Different zones of the growth plate. In the upcoming paragraphs, I will discuss the characteristics and the role of each of the growth plate zones in endochondral ossification.

I will also present studies (microarray analysis) to study the differential regulation of genes in these zones. The resting (reserve) zone: The resting zone consists of resting cartilage cells . The characteristic feature of these cells is that they are small, uniform, compactly located chondrocytes which are present as single cells or in pairs. These cells are rich in lipid and cytoplasmic vacuoles. Moreover, there is more extracellular matrix (ECM) than cells in this zone.

This zone is also characterized by having a low rate of proliferation and proteoglycan and collagen type IIB synthesis. The grove of Ranvieer is surrounding this zone (4). The role of the resting zone in endochondral ossification: There is an existing possible hypothesis that the resting zone contains chondrocytes (stem cell like cells) which acts as progenitor cells that are able to give rise to chondrocytes that are characterized by having rapid division. Each clone will constitute a cell column that is aligned parallel to the long axis of the bone.

When the cells undergo replication, and inorder to keep the columner organization, the two daughters line up parallel to the long axis (6). It is worth mentioning that there is different composition of collagen in different growth plate zones. Collagen Il is the major component of the resting and proliferative zone where as the collagen type X is present in the hypertrophic zone. The role of the resting zone in the endochondral ossification is summarized by three jobs: the resting zone is characterized by containing stem cell like cells which are able to give rise to clones of proliferating chondrocytes.

It is also suggested that the resting zone is able to generate an orienting factor (morphogen) of the growth plate which will play a role in the proper alignment of the proliferative clones into columns parallel to the long axis of the bone. Finally, an interesting possible role of the resting zone is its ability to produce a morphogene which will inhibit the terminal differentiation of the chondrocytes of the proliferative zone chondrocytes thus playing a role in the generation of different zones of the growth plate (proliferative and hypertrophic) (6). Proliferative zone:

Adjacent to the reserve zone is the zone of proliferation. This zone is exhibited by round chondrocytes that are actively proliferating. These cells form multicellular layers and hence become flattened (4). Role of proliferative zone in endochondral ossification: The proliferative cells will finally differentiate to form the hypertrophic chondrocytes that will consequently undergo apoptosis and contribute to ossification and formation new bone growth (8). As mentioned above, the chondrocytes in the proliferative zone secrete two major components of the ECM that is collagen Il and aggrecan.

Inorder to maintain the cells in continuous division ,there are several factors (endocrine and Indian hedgehog (1hh) and parathyroid hormone-related protein). The PTHrP is produced by chondrocytes that are present in the epiphyseal and perichondrium , and thus keep the chondrocytes in a proliferative state. The expression of B-cell leukemia-2 protein (Bcl-2) is induced by the PTHrP which inturn will inhibit apoptosis by blocking the pro apoptotic effects of Bcl-2associated X protein (BAX) (7).

In more details, the growth hormone GH has a significant role in maintaining the chondrocytes in a proliferative state. Growth hormone is produced by the pituitary glans and that exerts its effects on the growth plate through stimulation of secretion of insulin-like growth factor 1 (IGF1), both by liver cells and by growth plate chondrocytes (2). Hypertrophic zone: The proliferative cells of the proliferative zone will finally differentiate and enlarge to form the hypertrophic zone.

The hypertrophic chondrocytes at the end undergo apoptosis and contribute to ossification and new bone growth (8). The extracellular matrix of the cartilage is formed by the secretions (proteins and proteoglycans) of the chondrocytes. The hypertrophic zone is then occupied by blood vessels and differentiating osteoblasts and osteoclasts, which remodel the cartilage into bone tissue. All this process will result in the formation of new bone underneath the growth plate and hence bone elongation.

There is a tight regulation of the chondrocyte differentiation, proliferation, cartilage matrix secretion, cell death, and of vascular and bone cell by a series of paracrine signaling molecules. Among these factors is the insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), Indian hedgehog (IHH) and parathyroid hormone-related protein (PTHrP), bone morphogenic proteins (BMPs), WNTs, and vascular endothelial growth factors (VEGFs (10) As mentined above, the replacement of hypertrophic cartilage by the bone tissue is the result of the terminal step of hypertrophy of chondrocytes.

An essential role in the growth plate is played by the hypertrophic chondrocytes. That is it is not only about the increase in cell volume (hypertrophy) and longitudinal bone but these cells also act as signaling centers that secrete growth factors, cytokines, and other signaling molecules that can act on other cell types involved in endochondral ossification, such as osteoclasts, osteoblasts, and endothelial cells.

Hence, the process of differentiation and the action of chondrocytes is a highly regulated process by several systemic and local factors which include growth hormone, insulin-like growth factors (IGFs), thyroid hormone, parathyroid hormone-related peptide (PTHrP), Indian hedgehog, fibroblast growth factors (FGFs), canonical and noncanonical Wnt signaling, transforming growth factor-? (TGFB) family members, C-type natriuretic peptide, and others. Within the cell there is control of cartilage growth by classical mediators such as ? catenin in canonical Wnt signaling and Smad proteins in TGFf/bone morphogenetic protein signaling together with common kinases (e. g. , MAP and PI3K/Akt) and GTPases (e. g. , Rho GTPases). On the other hand, numerous molecular markers characterize the central stages of the chondrocyte life cycle. Chondrogenesis is typified by the expression of Sox transcription factors 5,6 and 9. Proliferating chondrocytes synthesize an ECM composed mainly of collagen II and aggrecan, among others, while the central ECM molecule expressed in hypertrophic cartilage is collagen X.

Factors expressed at the chondro-osseous junction regulate chondrocyte apoptosis and mineralization of the cartilaginous ECM. Late hypertrophic chondrocytes express factors that promote angiogenesis, bone deposition and the secretion of bone-specific cell ECM. These factors include VEGF (vascular endothelial growth factor), Mmp13 (matrix metalloproteinase 13), Mmp9 and Ibsp. Additional markers of the osteoblast and osteoclast phenotype, including core-binding factor alpha 1/ runt-related transcription factor 2 (Cbfa1/Runx2), acid phosphatase 5, tartrate resistant (Acp5) and tumor necrosis actor (ligand) superfamily, member 11 (Tnfsf11; RANKL/receptor activator of NF-kappaB ligand) are upregulated in hypertrophic cartilage and cells in the zone of ossification. The differential expression of genes between different zones by microarray studies on micro dissected tissue (summary): There are different techniques that are applied to study the differential gene expression in growth plate chondrocytes. These include immunohistochemistry, in situ hybridization using cell lines isolated early-stage limb mesenchyme, whole growth plates, or laser-capture microdissection.

The differential expression of genes in the different zones was studied. To summarize the results, it was found that several genes including cartilage genes, molecular chaperones, metabolic genes and genes involved in gluconeogenesis were enriched in zone II( proliferative zone)n. In zone III (hypertrophic zone), the genes that have molecules which are important in heparin binding, angiogenesis and chemokines, were identified. In zone III there was extensive enrichment gene sets for blood, phosphatases, cartilage and MAPK (11).

B) A commonly used apparatus to measure the effect of mechanical stress on the growth plates is the application of llizarovtype apparatus to the tails of rat. Dr. lan stokes with his colleagues (12), studied the effect of mechanical compression on the rat tail intervertebral discs. The rat tails were loaded with the apparatus described and the analysis included measurement of the disc thickness and angular laxity. The analysis involved the calculation of the water, proteoglycan and collagen contents.

In this studies the auther are trying to study the effect of modified mechanical loading conditions on the degeneration of the intervertebral disc, particulary effects on disc thickness, axial and angular stiffness and content of proetoglycans. Results of this study clarify that the thickness of the disc is changed in the compression group as well as decrease in the axial compliance and an increase in the angular laxity. Biochemical analysis of the compression group also show that the glycosaminoglycan content and the hydroxyproline in the discs was higher than control and sham.

The following results support the hypothesis that the mechanical compression cause changes in the mechanical properties and composition of rat tail discs. To understand the the effects of mechanical compression that is implicated in scoliosis , one of the studies was conducted by lan stockes to investigate the possibble effects of altered biomechanical environment, that was applied on rat tail, on the disc tissue specifically the effects on tissue synthesis and the gene expression patterns.

To study the impact of compression on the growth of 5 week old immature Sprague Dawely rats the discs of the rats was exposed to variable mechanical stress by using an external apparatus that applies loading and deformity for a period of 5 weeks . the experiments were performed in 4 rat groups, angulation group 15 degrees, angulation with Compression (0. 1 MPa), compression only 0. 1 MPa and reduced mobility in addition to the sham group and control. After 5 weeks, the changes in disc height and matrix compostion (water, DNA, GAG, and HA) was assessed and proline and sulphate incorporation and mRNA expression were measured at 5 days.

The results from experimental rats show that in these animals the disc space was diminished as compared to control group, however the levels of hydration and the amount of DNA was not affect between both groups. Intersetingly, the Glycosaminoglycans content was found to be different as well as a decrease in the size of hyaluronic acid . Analysis of the disc space in the experimental rats show that the disc space was diminished as compared to control group. The level of hydration and DNA content was not different between intervention and controls however, the GAG content was found to be different.

Effect of disc compression was reflected by a decrease in the hyaluronic acid size. Moreover, there was an increase in the incorporation of triated proline in the loaded rats but not the control yet no difference in sulfate incorporation was observed. Analysis of gene expression between different groups shows variation between one group to the others. For example, there was a reduction in the expression of Collagen-1 reduced ] goups A and B when compared to group C. There was also a reduction in TIMP-3 in group A but not in group C, and reduction in aggrecan from group B as compared to group C. inally, the levels of MMP-3 and TIMP-3 were reduced in group A compared to group R. There was clear reduction in the size of Hyalurinic acid (HA) and this was consistent with the modified disc remodeling after exposure to different types of loading and hence supports the fact that the compression is associated with HA depolymerization. Yet, its not clear if the observed change in HA size could affect disc function (13). In another study it was an attempt to study the relationships between the application of different stresses that would have an effect on growth plate.

It is well sited that the continuous application of mechanical stress would affect and modify the growth of long bones and hence affecting the progression of skeletal deformities during growth. In this study the authors used non human growth plates to quantify the modifed growth in reponse to exposure to different magnitudes of maintained altered stress.. Figure 2: Ilizarovtype apparatus on the rat tail. Commonly used method to study the effect of mechanical compression on the disc is the load of rat tails with Ilizarovtype apparatus.

For analysis, the effect on the sham group was substracted, and the sensitivity of the growth to stress is averaged 17. 1% per 0. 1 MPa and the relationship between the the growth rate and the amplitude of the mechanical stress applied is represented in the following formula: where G is the actual growth, Gm is the mean baseline growth (unaltered stress), s is the actual stress on growth plate (compression negative), and sm is the mean prevailing (baseline) stress on growth plate.

It appears that there is a linear relationship between effect of stress and the ratio of change in growth. In the tested animals, it was obvious that distraction accelerated growth however compression slowed growth. in more details, when the compressive stress was doubled, approximately the growth rate was reduced in doublet. The Hueter-Volkman principle, explains that the deformity progression in skeletal disease such as scoliosis depends on biomechanical effect. The results of the present study with consistent growth modulation effect measured in nonhuman species.

These findings could be extrapolated to human disease in order to study the expected response of any human growth plate to a specific stress level (14). Inorder to investigate the effect of severly applied compressive forms on the mechanical properties and composition of rat tail intervertebral discs, this question was addressed. The hypothesis suggests that when the discs of the rat are loaded with compression force , this will reduce the thickness of the disc, will also affect the axial and angular stiffness which will be increased and the content of proteoglycans is decreased.

It was found in this study That in experimental animals the level of glycosaminoglycans (GAG) was significantly upregulated .. There were no significant effects of disc level on water or hydroxyproline contents. gly-cosaminoglycan and hydroxyproline quantities in the discs in the compression group were significantly greater than in the discs in the sham and immobilization groups. The above results indicate that the mechanical properties and composition of tail discs is changed in response to the exposure of severe mechanical stress (14).

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