Abstract

Bones are formed through a process known as osteogenesis from three different lineages. This process takes place through two different methods, which are intramembranous ossification and endochondral ossification. A large increment in bone mass takes place in childhood, as well as puberty, through the formation of endochondral bone. At this point, growth hormones are known to play a role. This paper will focus on the process of cartilage and bone formation and the stimulation of bone formation by the growth hormone. There are a number of factors that regulate the formation of bone, while growth hormone affects a number of factors related to bone formation including longitudinal bone growth, longitudinal cartilage growth, osteoblasts, and osteoclasts. The somatomedin hormones are produced after being stimulated by the somatotropin hormone (STH) in order to promote cell division and growth. A number of hormones are essential in the normal growth of postnatal longitudinal bone, though it is usually accepted that growth hormone is the most crucial hormone in this regard. Moreover, it has been shown that growth hormone induces growth of cartilage as well as other tissues through raising the number of cells rather than through raising the size of the cell.

Introduction

Bone, which is an extremely specialized framework that supports the body, is qualified by its hardness, power of repair and regeneration, as well as rigidity. It offers protection to the vital organs and offers a marrow environment. It also serves as a mineral reservoir for homeostasis of calcium, as well as cytokines and growth factors reservoir. It, as well participates in balance of acid and base [1]. Bone invariably goes through modeling during life in order to aid in adapting to modification of biomechanical forces and remodeling in order to get rid of old, micro-destroyed bone and its replacement with new, stronger bone that aid in the preservation of strength of the bone.
Osteogenesis is the process through which bones are formed, from three different lineages, which occurs by means of two different methods, namely intramembranous ossification and endochondral ossification [2]. Intramembranous ossification refers to the characteristic way through which the turtle shell and the flat bones making the skull are formed. On the other hand, endochondral ossification is the method that is responsible for the formation of embryonic bones and growth in length. A large increment in bone mass takes place in childhood, as well as puberty, through the formation of endochondral bone. Here, growth hormones are recognized to play a role. There are a number of hormones are essential in the normal growth of postnatal longitudinal bone, though it is usually accepted that growth hormone is the most crucial hormone in this regard. Growth hormones induce growth of longitudinal bone directly and raise the local generation of IGF-I through stimulation of transcription of the IGF-I gene resulted in the suggestion that the dual effector theory of action of growth hormone is valid for the control of growth of longitudinal bone, as well. This paper is aimed at focusing on the process of bone and cartilage formation, as well as the role of the growth hormone in stimulation of bone formation.

Intramembranous Ossification

Intramembranous ossification refers to the characteristic way through which the turtle shell and the flat bones making the skull are formed. In the process of making the skull through intramembranous ossification, the mesenchymal cells that are derived from neural crest proliferate and condense forming compact nodules. A number of these cells form capillaries while others undergo changes in their shape becoming osteoblasts, which are the committed precursor cells in bone formation. The collagen-proteoglycan matrix, which is secreted by osteoblasts, has the capability of binding calcium salts. This binding enables the prebone (osteoid) matrix to get calcified. More often, osteoblasts undergo separation from the region that has been calcification by an osteoid matrix layer they secrete (Figure 1). On rare occasions, osteoblasts are entrapped in the matrix that is calcified to become osteocytes, which are the cells that make up the bones. As the process of calcification continues, bony spicules spread out from the area where ossification started. Furthermore, the whole area of calcified spicules is encircled by compact mesenchymal cells, which form the periosteum. The periosteum is the membrane surrounding the bone. The cells that are located on the inner surface of the periosteum are also converted to osteoblasts and are involved in depositing osteoid matrix parallel to the one of the existing spicules. This results in the formation of many bone layers [2].
The mechanism through which intramembranous ossification occurs involves morphogenetic proteins of the bone and the activating transcription factor known as CBFA1. The bone morphogenetic proteins such as BMP2, BMP3, and BMP4, which come from the head epidermis are considered to be the ones that instruct mesenchymal cells that are derived from the neural crest to directly become the bone cells. The BMPs proteins are involved in activating the Cbfa1 gene located in the mesenchymal cells. The CBFA1 transcription factors have the capability to transform mesenchymal cells making osteoblasts [3]. It has been found that the mRNA produced by CBFA1 in the mouse is severely restricted to the condensation of mesenchymal cells that form bone and at the same time limited to the osteoblast lineage. It is this protein that activates the genes which produce osteopontin, osteocalcin, as well as other extracellular matrix proteins that are bone specific [2].
The intramembranous ossification is involved in the formation of flat bones that make up the skull and mandible. This process has no cartilaginous precursor, and the formation of the bones occur directly. The osteoblasts produce osteoid that are proteoglycans and collagen and lead to bone mineralization and the formation of spicule. Spicules are involved in the production of spongy bones. The growth of bone may be described to be an appositional growth where the bone is laid down on the outer surface of the bone that is developing. In the course of bone development, there is a rise in the association between blood vessels and the formation of bone marrow by the mesenchyme cell, between the blood vessels and the bone [2].

Endochondral Ossification

Endochondral ossification is the second method through which bones are formed. This method is the one that is responsible for the formation of embryonic bones and growth in length [2]. The endochondral ossification occurs through a cartilaginous precursor. The process starts with the condensation of mesenchyme in order to form the bone outline. The high cell density causes a trigger to the core cells causing them to differentiate forming chondrocytes and starts cartilage matrix secretion. The proliferation process is continued, and the shape of the bone is formed with shaft or diaphysis and epiphysis or bulbous ends. The chondrocytes located in the middle of the diaphysis are the ones that stop proliferating resulting to hypertrophy. A restrictive membrane known as perichondrium surrounds the shaft, and this means that the bone only has a chance of growing in length and not width. There is also pushing away of the epiphyses from each other. The chondrocytes die once they have gone through hypertrophy and the osteoblasts that are on the interior side of the perichondrium attack the cartilage matrix after which they release the extra-cellular matrix. The release of extra-cellular matrix is one of the characteristics of the bone. The process of ossification takes place from the outer side toward the inside, and this is referred to as the primary or perichondrial ossification. There is an invasion of the diaphysis central region by the blood vessels in order to transport osteoclasts as well as bone marrow stem cells [4].
Lack of perichondrium that surrounds the epiphysis allows for the length of the bone to be expanded. However, this lack is an indication that there is lack of surrounding osteoblasts that can be invaded. In order to accomplish osteogenesis, blood vessels do their invasion from the diaphysis carrying osteoblasts. In the epiphysis, there is the initiation of bone growth by the osteoblasts from outside towards the inside of the bone, and this is known as secondary ossification. Secondary ossification, in some animals, take place after birth and the bones are covered in a layer that contains osteoblasts [5].
In mammals, the cartilage model that makes up the skeleton is only seen during the early development stages. The cartilage is later replaced by bones although cartilages in specific body [6]. These places where cartilage does not undergo replacement are the area between the epiphysis and diaphysis, which is known as the epiphyseal growth plate (EGP) and at the end areas of epiphysis, which are between the joints and acts shock absorbers.
The process of endochondral ossification is mainly involved in cartilage formation from the mesenchymal cells that are aggregated and subsequently replacing the cartilage tissues with bone. The endochondral ossification process is divided into five major stages. In the first stage, there is committing the mesenchymal cells to make cartilage cells. The commitment occurs as a result of paracrine factors, which induce the mesodermal cells that are located near causing them to express transcription factors, Scleraxis and Pax1. The two transcription factors are believed to be involved in the activation of the cartilage-specific genes. The scleraxis transcription factor is expressed from sclerotome located in the facial and limb mesenchyme forming the cartilaginous precursors that form to bone [7].
In the second phase of the process of endochondral ossification, the mesenchyme cells that have been committed condense forming compact nodules and then differentiating into chondrocytes, which are the cartilage cells. The N-cadherin is seen as an essential substance in the initiation of condensations while N-CAM appears to be essential in maintaining the condensation. The SOX9 gene that encodes for a DNA-binding protein in human is expressed before cartilaginous condensations. Presence of mutations in the SOX9 gene results to camptomelic dysplasia, which is a rare disorder in the development of the skeleton. This leads to the deformities of many bones in the body. Most babies with SOX9 gene may die from respiratory failure as a result of the tracheal and rib cartilages that are poorly formed [8].
In the third phase of the process of endochondral ossification, there is a rapid proliferation of the chondrocytes forming bone models. As the chondrocytes divide, there is secretion of an extracellular matrix, which is cartilage-specific. In the fourth phase, chondrocytes stop their division process and their volume increase dramatically and thus they become hypertrophic chondrocytes. The enlarged chondrocytes change the matrix that they produce through the addition of collagen X and additional fibronectin. The addition enables the matrix to be mineralized by calcium carbonate [9].
In the fifth phase, there is the encroachment of the cartilage framework by blood vessels. The death of hypertrophic chondrocytes occurs through apoptosis creating a space that becomes the bone marrow. In the process of cartilage cell death, the group of cells surrounding the cartilage model undergoes differentiation to form osteoblasts. The formed osteoblasts start the formation of bone matrix on the cartilage that is partially degraded. In the end, all the cartilages are replaced with bone cells and, therefore, cartilage tissues work as a model that the bones can follow. Some of the bones whose skeletal components start from cartilage and later change to bones include the pelvis, vertebral column, and the limbs.
The process of chondrocyte replacement by bone cells mainly depends on the extracellular matrix mineralization. This process is illustrated in a clear manner through skeletal development of the chick embryo that makes use of the calcium carbonate using eggshell as the source of calcium. In the process of its development, chick embryo through its circulatory system translocates approximately 120 mg of calcium derived from the eggshell and taken to the skeleton. Removing the chick embryo from their shell after day 3 and transferred in a culture that has no shell for the whole time of their development, there was no maturation of the cartilaginous skeleton into bony tissue.
In the process of adding new bone material from the internal periosteum surface, the internal region is hollowed out forming the bone marrow cavity. This form of bone destruction is performed by the osteoclast, which are multinucleated cells entering the bone via the blood vessels. The osteoclast cells are probably generated from same precursors just like the macrophages. They dissolve protein components of the bone matrix, as well as the inorganic components. Each osteoclast offers extension to several processes into the matrix while pumping out hydrogen ions from the matrix onto the neighboring material. This leads to the acidification of the material and thus solubilizing it. Once the bone marrow is formed, the blood vessels bring in the blood-forming cells, which stay in the marrow in a life time. The number of osteoclasts, as well as their activity, should be regulated tightly to avoid osteoporosis when the activity or number of osteoclasts is high or osteopetrosis when the activity or the number of osteoblasts is low [10].

Regulation of Bone Formation

There are various discoveries through human and murine mutations that result in abnormal development of the skeletal structure that have shown that differentiation, patterning as well as the proliferation of the chondrocytes, are tightly regulated. Regulation may occur through the growth factor receptors, insulin-like growth factors, estrogen receptors, and extracellular matrix proteins. Presence of the fibroblast growth factors has been shown to stop the proliferation of the facial cartilage and epiphyseal growth plate cells [11]. These factors instruct the precursors of cartilage to differentiate instead of dividing. The epiphyseal growth plate cells have been shown to be very responsive to various hormones, and the proliferation of these cells is usually stimulated by the insulin-like growth hormones and growth hormones. Studies have shown that insulin-like growth factor I or IGF-I production in the epiphyseal chondrocytes is stimulated by growth hormones. The chondrocytes then respond to the IGF-I production by proliferating. Combining IGF-I and growth hormone provide a very strong mitotic signal showing that IGF-I is necessary for the normal growth shoot at puberty.
The estrogen receptors are also involved in the maturation of cartilage. Several sex hormones are involved in the induction of pubertal growth spurt as well as the subsequent growth cessation [12]. When puberty is over, the increased levels of testosterone or estrogen lead to the epiphyseal plate cartilage that is remaining to undergo hypertrophy. The cartilage cells may continue to grow, die or be replaced to form bones. When there is no more cartilage formation, there is no more growth of these bones and this lead to a process called growth plate closure. Other hormones that are involved in the regulation of hypertrophy program and maturation of the epiphyseal growth plate. This explains why those children suffering from hypothyroidism are in danger of developing disorders of the growth plate [13].

Effect of Growth Hormones

Growth hormone affects a number of tissues which include muscle, liver, bone, as well as kidney. Effects of growth hormone on kidney and muscle have lately been comprehensively reviewed in Endocrine Reviews by Feld and Hirschberg and Florini et al. A large increment in bone mass takes place in childhood, as well as puberty, through the formation of endochondral bone. A slow increment in bone mass is subsequently seen until there is the attainment of peak bone mass at 20–30 years of age. Subsequently, decreases in bone mass with a sped up the loss of bone observed in females following menopause. Remodeling of bone is modulated by a balance between formation of bone and resorption of bone. In this process, growth hormone is recognized to play a role. A total gain of skeletal mass because of the formation of new bone resulting growth hormone was first demonstrated in adult mongrel dogs. Following treatment with growth hormone for three months a 2% rise in cortical bone mass, as appraised by histomorphometry, was discovered.
Because of restrictions in the supply of growth hormone, a confined number of the animal as well as clinical investigations were carried out until the mid-1980s when recombinant human growth hormone became available. The first application of recombinant human growth hormone was limited to treatment of growth-retarded growth hormone-deficient children. Nevertheless, it is currently well established that growth hormone also exercises significant effects in grownups, and growth hormone treatment of growth hormone-deficient grownups is now sanctioned in a number of nations. Recent investigations, in both human beings and animals, have shown that growth hormone exerts strong effects on remodeling of bone [14].

Effects of Growth Hormone on Longitudinal Bone Growth

In the process of growth of longitudinal bone prechondrocytes in the layer of germinal cell differentiate and thenceforth go through restricted clonal enlargement in individual columns of chondrocyte, in the growth plate. Later on, cells in the hypertrophic area mature as well as degenerate are finally included into bone [15]. A number of hormones are essential in the normal growth of postnatal longitudinal bone, though it is usually accepted that growth hormone is the most crucial hormone in this regard. Moreover, it has been shown that growth hormone induces growth of cartilage as well as other tissues through raising the number of cells rather than through raising the size of the cell. A broadly debated question in the course of the last several years has been whether growth hormone acts directly on tissues, or whether the result is mediated by a growth factor that is derived from the liver, at first named sulfation factor, but afterward renamed somatomedin, and then demonstrated to be identical to IGF-I. Based on the original hypothesis of somatomedin, growth hormone induces skeletal growth through stimulation of production of somatomedin by the liver which, successively, induces growth of longitudinal bone in an endocrine way [16].

Effects of Growth Hormone on Longitudinal Cartilage Growth

Growth of longitudinal bone results from new cells recruitment from the layer of stem cell and the subsequent increase of the cells that are differentiating in the long bones growth plate. In growing persons, the rate of cartilage cell multiplication is at equilibrium with the rate of calcification of cells at the plate’s diaphyseal end. Hence, the breadth of the growth plate is about the same even though the hoarded length of the bone rises. As new cells begin their differentiation programme, and go through a restricted clonal enlargement, the cells within the growth plate are incessantly renewed up to the time of epiphyseal closure during sexual maturation. Of the endocrine aspects that regulate growth of skeleton, growth hormone is the single acknowledged hormone, which induces growth of longitudinal bone in a manner that is dose-dependent over a broad range of doses. This fact makes the ground for the in vivo assay referred to as the tibia test in which the breadth of the tibial growth plate is ascertained by a histomorphometric method.
Difficulties in showing in vitro consequences of growth hormone on a number of growth-related parameters, for instance incorporation of thymidine and uptake of sulphate in costal cartilage of hypophysectomized rats in mixture with the discovering that adding serum from normal but not hypophysectomized rats caused stimulation of these parameters, made the ground for the somatomedin hypothesis of action of growth hormone. Based on this theory, growth hormone induced growth indirectly through stimulation of the generation of factors of serum, which mediated the growth-enhancing effects of growth hormone. The somatomedin hypothesis was then reinforced by the keying out of insulin-like growth factor-I as the main serum somatomedin as well as the observation that growth hormone induced the production together with the release, from the liver, of this peptide.
A number of recent studies have demonstrated that local administration of growth hormone at the location of the epiphyseal growth plate of hypophysectomized rats induces unilateral growth of bone. It has also been shown that infusion of growth hormone into one of the femoral arteries of hypophysectomized rats induces growth of longitudinal bone of the treated leg, providing strong prove for the hypothesis that growth hormone induces the differentiation as well as the proliferation of a number of, as yet unidentified, epiphyseal chondrocytes [17]. Schlechter et al had the crucial observation that the stimulatory consequence of locally administered growth hormone on growth of longitudinal bone was totally gotten rid of if the antiserum to IGF-I was co-infused with the hormone, proposing that the stimulatory consequence of growth hormone on growth of longitudinal bone was reliant on the presence of endocrine or locally produced (paracrine/action) IGF-I.
Immunohistochemistry by use of monoclonal antibodies against the receptor of growth hormone has disclosed receptors in the proliferative, hypertrophic as well as germinative zones of sagittal segments of the proximal tibia growth plate of a rabbit, offering a structural ground for direct interaction between growth hormone and epiphyseal chondrocytes at a number of maturational phases. Receptors of growth hormone have as well been visualized in rabbit and rat cultured epiphyseal chondrocytes. It has, recently, been shown that epiphyseal chondrocytes in monolayer culture show mRNA of growth hormone receptor and that this expression is partly controlled by growth hormone offering prove for a direct interaction between growth hormone and epiphyseal condrocytes [15].
Through studying the effects of IGF-I and growth hormone in 3T3 preadipocytes, Green and his colleagues had the observation that IGF-I and growth hormone act on cells at varied stages of maturation. Therefore, growth hormone was discovered to induce young preadipocytes while IGF-I induced cells at a later phase of development. The hypothesis by Green and his colleagues, that growth hormone acts on progenitor cells and that IGF-I induces the subsequent clonal enlargement, was tagged the dual effector theory.
The results that growth hormone induce growth of longitudinal bone directly and raises the local generation of IGF-I through stimulation of transcription of the IGF-I gene resulted in the suggestion that the dual effector theory of action of growth hormone is valid for the control of growth of longitudinal bone, as well. Subsequent in vitro investigations by use of cultured epiphyseal chondrocytes in suspension disclosed that growth hormone and IGF-I induce cells at different phases of maturation. Therefore, growth hormone induces the formation of a colony of young prechondrocytes while IGF-I induces cells at a later phase of maturation, providing back up to the theory that maturation of cells is indeed a crucial factor ascertaining responsiveness of epiphyseal chondrocytes to growth hormone, as well as IGF-I [16].

Effects of Growth Hormone on osteoblasts

There have been studies of growth hormone effect in several osteoblastic cell lines as well as primary isolated cells of several sources, including chicken, human, mouse primary cells and rat, and, the UMR 106.01 rat osteosarcoma and. SaOS-2 human cell line. Growth hormone stimulates proliferation of primary isolated rat, human, mouse, chicken, and osteosarcoma cells of a rat, as well as cells from rat cell line that is osteoblast-like and osteosarcoma cells of humans. The effective growth hormone concentrations are in the physiological range, proposing that growth hormone exerts direct actions on osteoblasts [18].

Effects of Growth Hormone on osteoclasts

Growth hormone raises the number of osteoclasts in the proximal tibia’s metaphyseal bone of hypophysectomized rats. Nevertheless, this effect’s mechanism is less clear. Growth hormone receptor mRNA has been observed in cultures of mouse marrow as well as in hemopoietic blast cells of mouse. In a study done recently by Nishiyama et al [19] by use of hemopoietic blast cells and stromal cells of mouse, it was discovered that growth hormone triggers resorption of osteoclastic bone via direct, as well as indirect actions on indirect activation and osteoclast differentiation of mature osteoclasts. Aspects, which may enhance the indirect formation of growth hormone-regulated osteoclast, include IL-6 and IGF-I, both of which participate in the formation of osteoclast and have been demonstrated to be controlled by growth hormone [20]. It has previously been shown that IGF-I backs formation and activation of osteoclasts in unfractionated mouse bone cells cultures (Ransjo, Lerner and Ohlsson)and that osteoblasts mediate IGF-I-stimulated osteoclasts formation in cultures of mouse marrow as well as isolated rat osteoclasts activation. Moreover, functional IGF-I receptors are expressed by human osteoclasts. In another study by Ransjö et al. [21], by use of marrow cultures of mouse, growth hormone resulted in suppression of formation of osteoclast by a mechanism that is IGF-I-independent.
In vivo models of animals are useful when assessing the influence that treatment with growth hormone on bone mass changes, mechanical strength of bones, as well as bone metabolism. For histological studies, the models are excellent since there is a possibility to carry out dynamic and static histomorphometry and to assess differences in regional reaction. Administration of systemic growth hormone raises other circulating hormones levels, which influence the active vitamin D metabolite and bone such as IGF-I.
Until now, administration of systemic growth hormone has been applied in almost every animal experiment, and it has been not possible to clarify if the measured modifications are as a result of systemic or local stimulation of growth hormone. Growth hormone receptors have been observed in femur epiphyseal of rat and calvarial osteoblasts by use of immunoreactive as well as mRNA techniques [22]. The local GH delivery effect to a bone has been analyzed in rats, and the local GH expression effects in GH-transgenic mice bone tissue have been demonstrated. These recent in vivo studies demonstrate that growth hormone has the ability to trigger the formation of via direct interaction with bone tissue.
The growth hormone receptor was the first keyed out member of the superfamily of cytokine receptor [23]. Other members of the class 1 superfamily of cytokine receptor are erythropoietin, prolactin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, ciliary neurotrophic factor, leptin, thrombopoietin, and interleukins. The superfamily also includes subunits of receptors, which interact with over one cytokine receptor. The growth hormone-binding protein is the circulating form of growth hormone receptor that is soluble. It is differentially brought forth among species, for instance, optional splicing in rat and mice leading to the addition of a carboxyl terminal sequence that is hydrophilic proteolysis of the receptor that is membrane-bound in other species. The growth hormone binding protein is thus identical to the receptor’s extracellular domain when produced through proteolysis. A number of short, but growth hormone receptor’s membrane-anchored isoforms have been depicted in human beings (Ross et al., 1997). Because of the lack of activity of intrinsic kinase cytokine receptor, superfamily members recruit or activate cytoplasmic tyrosine kinases to relay their cellular signals [24]. The cytokine receptor superfamily members are generally thought to elicit their signaling through the use of nonreceptor tyrosine kinases like Src and/or JAK family kinases. Binding of GH to the receptors that are on the surface of the cell induces receptor dimerization with a subsequent association, as well as activation of JAK2. Subsequently, other key groups signaling molecules such as EGF receptors and kinases like c-Fyn, c-Src, and FAK, as well as MAPK, IRS group members are all activated (Figure 2) [25].

Conclusion

In conclusion, the somatomedin hormones are produced after being stimulated by the STH in order to promote cell division and growth. Somatotropin, which is a polypeptide growth hormone in human, is released by the anterior pituitary gland and is also referred to as human growth hormone. The somatomedin is the one that mediates the effect that is caused by somatotropin. Production of somatomedins occurs in various body tissues such as the liver, and their action may be paracrine or autocrine. The somatomedins are also involved in the production of somatostatin, which suppresses the release of growth hormone. Therefore, the level of growth hormone and somatomedin are under a negative feedback. Increasing the level of samatomedin reduces the level of growth hormone through the stimulation of somatostatin production. A number of hormones are essential in the normal growth of postnatal longitudinal bone, though it is usually accepted that growth hormone is the most crucial hormone in this regard. Moreover, it has been shown that growth hormone induces growth of cartilage as well as other tissues through raising the number of cells rather than through raising the size of the cell.
Figure 1: Intramembranous ossification schematic diagram showing mesenchymal cells that have been condensed to give osteoblasts that deposit osteoid matrix [2].
Figure 2: The mechanism of growth hormone signal transduction [25]

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