Bone Development and Growth

Abstract

Osteogenesis is a complex process of bone formation involving several phases and utilizes various cell, metabolites, hormones, and organic and inorganics components. Numerous genetic factors mediate bone formation. Initially, progenitor cells produce osteoblastic lines, which pass through three major cell differentiation stages: proliferation, maturation of matrix, and mineralization. Based on embryonic origin, ossification is of two types: intramembranous and endochondral. In intramembranous ossification, mesenchymal cells in ossification center directly differentiate into osteoblasts, without prior cartilage formation. It involves mesenchymal cell proliferation in highly vascularized areas of embryonic connective tissue, leading to primary ossification center formation. These cells then synthesize bone matrix at periphery, with continuous differentiation into osteoblasts. The resulting bone undergoes reshaping and is eventually replaced by mature lamellar bone. Sufficient blood supply and communication among cells by lacunar-canalicular system are crucial for bone synthesis and maintenance. In contrast, endochondral ossification begins with the formation of primary ossification center within cartilage. Chondrocytes undergo proliferation, expanding the cartilage through cartilage matrix deposition. Central region of cartilage sees the maturation of chondrocytes into hypertrophic chondrocytes. As primary ossification center forms, marrow cavity expands toward epiphysis. The process is completed by subsequent stages of endochondral ossification in various zones of ossification.

1. Introduction

Bone is the toughest connective tissue in the human body, consisting of 50% water and other solid elements that involve several minerals. Specifically, 33% of cellular material is made up of 76% calcium salt. The bone’s primary source of nutrition throughout development is the blood supply, and hormones play a fundamental role in regulating this process. Osteoblasts, osteoclasts, and other bone-forming cells are responsible for determining new bone growth [1].

Bone serves three major purpose: firstly, it plays a crucial role in maintaining calcium homeostasis and works as a storehouse of phosphate (PO₄³⁻), potassium (K⁺), magnesium (Mg⁺), and bicarbonate (H1CO₃⁻). Secondly, it provides protection to internal organs and mechanical support to soft tissues and acts as a lever for muscle contraction. This supports bodily functions, including mobility. And lastly, it is the primary site of hematopoiesis in adults [2].

The bone matrix is different from matrices of other connective tissue in regards that it regenerates continuously throughout the life because of bone turnover and physiological mineralization. Bone tissue has mineral as well as non-mineral constituents. On (and inside) the bone tissue, there are three different types of cells: (a) the osteoblasts that build new bone, which transform into (b) osteocytes when osteoblasts absorb mineral and (c) the osteoclasts that destroy or break down new bone. These cells communicate and interact with each another through signaling substance or direct cell contact [3].

1.1 Bone cells

The primary functions of bone cells, originating from the mesenchymal stem (MSCs) and hematopoietic stem cell (HSCs) lines, are the absorption of bone and remodeling of bone. Bone cells are divided into many categories according to their appearance, function, and distinct location [4]. A mature osteoblast has an enormous quantity of RER and huge Golgi apparatus. Some of the osteoblasts possess cytoplasmic projections in order to travel in bone matrix direction and finally reached at osteocyte process. At this stage, this OB has two choices: either they convert into osteocyte or BLCs or undergo programmed cell death. Surprisingly in the vacuoles of osteoblasts, there are some ovoid bodies having dense bodies, and some TUNEL-positive structures are found and seen. These findings indicate that they are also able to engulf apoptotic materials in addition to skilled phagocytosis during the alveolar development of bone (Figure 1) [2].

Figure 1.

Classification of bone cells as osteoblasts, osteoclast bone-lining cells, and osteocytes on the basis of their source, resorption, development, and functions. Mature osteoblast structure as a single cuboidal cell layer having enormous quantity of RER and huge Golgi apparatus.

1.2 Osteoblasts

The main cells responsible for forming bones are known as osteoblasts (OBs). These cells are in cuboidal and plump-like appearance and are organized in different layers to form matrix which is then mineralized extracellularly. Osteoblasts comes from mesenchymal stem cells (MSCs) that produce tendon fibroblasts, fat, muscle, or chondrocytes, as well as other types of fibroblasts [5].

For MSCs, to commit osteo-progenitor lineage, several genes must be expressed. This includes components of Wnt pathway and bone morphogenetic protein (BMP) synthesis. Osteoblast differentiation is basically based on expressions and activation of RUNX2, osteorix (Osx), and Dlx5. Among them, RUNX is considered more important for their differentiation because it upregulates ColIA1, BSP, and BGLAP as well as ALP and OCN (Figure 2).

Figure 2.

Osteoblast transformation after the bone-forming phase, the sealing zone, and ruffle border and ionic transport.

After the expression of ColIA1 and RUNX2, there starts a proliferation phase. At this stage, precursors of osteoblasts are referred to as pre-osteoblasts because they show alkaline phosphatase (ALP) activity.

Some transitions indicate the conversion of pre-osteoblast to mature OBs. This includes elevation of Osx synthesis and release of some bone matrix proteins. Additionally, configuration of osteoblasts changes, converting into large cuboidal-cells.

Other elements such as connexin 43, microRNAs, and fibroblast growth factor (FGF) are crucial for the differentiation of osteoblasts.

After the matrix formation, a new phase starts having two sub-stages, that is, vesicular and fibrillary stage in which matrix mineralization occurs. Organic matrix deposition and their mineralization are the two major steps for bone matrix formation through osteoblasts. During the initial stage, bone matrix is formed by the release of collagen type 1, non-collagenous proteins such as OCN, osteopontin, and BSPII, and proteoglycans such as biglycan or decorin.

After the matrix formation, a new phase starts having two sub-stages, that is, vesicular and fibrillary stage in which matrix mineralization occurs. Matrix vesicle releases from domains of membrane and connects to other substances as well as proteoglycans (PGs) in the vesicular phase. Ca²⁺ immobilizes these PGs. These Ca²⁺ release through some channels which are created by annexin protein when there is disintegration of PGs [6].

1.3 Bone lining cells

In contrast to osteoblasts that are present on the bone surface, these are some flat or thin barely noticeable cells which surrounds the bony inactive surface of the human skeleton. Initially, bone lining cells (BLCs) were considered to be preosteoblasts. Currently, it is accepted that osteoblasts that fail to suffer apoptosis and fail to differentiate into osteocytes become bone lining cells [7].

Although BLCs are present on many bone surfaces, but they are well-characterized on endosteal and endocortical surfaces. The majority of the skeletal surface is covered with BLCs and is inactive. Only a small percentage of the overall skeletal surface is remodeling at any one moment. Whereas, BLCs cover the non-remodeling or “inactive” bone surfaces. It is likely that these surfaces, together with the cells connected to them, are physiologically functioning in terms of mineral balance and calcium exchange. BLCs are flattened across the outside of the bone such that they seem exceedingly thin and elongated when sliced perpendicular to the surface. By light microscopy, it can be hard to differentiate BLCs from diverse cell types, including adventitial, stromal, marrow sac, and osteoprogenitor cells. Through gap junctions, they establish connections with neighboring bone lining cells (BLCs) and send cell processes into surface canaliculi.

Another ability that BLCs have is that they can differentiate into osteogenic cells; that is why, they act as a determined osteogenic precursor’s source. Along with other endosteal tissue cells, BLCs may do some crucial job in hematopoiesis and may be a key element of the marrow stromal system, possibly by regulating the inductive microenvironment. BLCs may play a role in the initiation of bone resorption and remodeling by transmitting the activation signal. Also, evidence illustrates the importance of BLCs in maintaining bone fluids and ion fluxes between interstitial fluid compartments and bone fluids for mineral homeostasis [8]. BLCs show intercellular adhesion molecule 1 while it do not express osteocalcin. These are two important phenotypic differences between BLC and osteoblasts.

1.4 Osteocytes

Osteocytes form more than 90–95% of all the bony cells, whereas osteoblasts and osteoclasts comprise about 4–6% and 1–2% of bone cells, respectively, in an adult skeleton. These are sporadically distributed along the whole mineralized matrix. Their cell body is encapsulated in a structure called lacuna and is connected with each other via dendritic process which traverses bone in a microscopic canal-like structure called canaliculi having dimensions of 250–300 nm [9].

Osteocytes display a distinct morphology both in vivo and in culture that is determined via the expression of E11/PDPN/GP38, PLS3, or CD44. These genes are also expressed in neurons. Osteocytes are now considered as the mechanosensory cells. They have a long lifespan and are found throughout the volume of the bone. Osteocytes give rise to proteins like sclerostin and RANKL, and OPG may influence on other bony cells via autocrine or paracrine pathways. All these happen because of hormonal and mechanical signaling [10].

1.5 Osteocyte formation

Osteocytes arise through the differentiation of osteoblast from the lineage of mesenchymal cells. Four different phases of this process involving (a) osteoid-osteocyte, (b) pre-osteocyte, (c) young osteocyte, and (d) mature osteocyte have been proposed. When the cycle of bone synthesis ends, some osteoblasts’ group may be converted into osteocyte embodied in the matrix of bone.

During this process, some notable configurational changes also occur, and this may include reduction in protein production or synthesis and also decrease in round osteoblast shape, which may be correlated with decrease in some organelle count [6].

Furthermore, these cells create a network among them by connecting with nearby osteocytes through many different kinds of lengthy procedures. The network called the lacunar-canalicular network promotes the exchange of nutrients and waste products between osteocytes and acts as a gap junction for communication. Their cell body is deeply present in lacuna, and with the help of canaliculi, their processes arrives to the surrounding osteocytes, and its supply is maintained by Haversian canals through which small vessels move (Figure 3) [7].

Figure 3.

Osteocyte with its lacuna, a structure containing osteocyte and canaliculi which connect one cell with another through various channels and allow them to exchange their nutrients and transmission of signals necessary for remodeling and repair of bone.

These cells serve as mechanosensors by means of this system as their connected network can detect mechanical loads applied on bone. This enables the bone to withstand stresses of daily life. Osteocytes also regulate remodeling by controlling the functions of osteoblasts and osteoclasts. Furthermore, it has been established that osteocyte death acts as a stimulus of bone resorption via osteoclasts [6].

Osteocytes detect microfractures in the mineralized bone that result from the mechanical force placed on the bone during movement. In response to this, the cells activate the remodeling process that fix the damaged bone. Osteocytes also detect metabolic signals in addition to the mechanical ones. Aging and estrogen withdrawal, which are linked with increase in bone remodeling and decreased bone mass, increase the risk of their death. Osteocytes in two ways affect osteoblasts: first, they upregulate osteoblasts by producing messengers such as prostaglandin E2 and nitric oxide, and second, they downregulate osteoblasts by releasing sclerostin [11].

1.6 Osteoclasts

The main cells which are involved in the breakdown of bones are osteoclasts. This role is essential for the maintenance, remodeling, and repair of the vertebral skeleton bones. Human osteoclast gigantic multinucleated entity is 150–200 μm in diameter and typically comprises four nuclei. The transformation of macrophages to osteoclasts by osteoclast-inducing cytokines leads to very massive cells, up to 100 μm in diameter. They may contain dozens of nuclei and express typically major osteoclast proteins, but because of the non-natural substrate, they differ greatly from the other cells that live in bone. The multinucleated constructed osteoclast’s size allows it to focus many macrophages’ vesicular, protein secretory, and ion transport capacities on a specific region of the bone. The bone multicellular units (BMU) that reshape the bone involves combined activities of osteoblasts, that build bone, and osteoclasts, that break down bone [12]. When osteoclast activity is dysregulated, bony mass rises. Osteoclast precursors stick on osteons’ surface and continue to divide, differentiate, and fuse to mature polynucleated entities to initiate bone remodeling. In order that the osteoclast adheres to the bony surface, it disintegrates the matrix loaded with minerals and runs across bony surface and causes certain active domain development. Each osteoclast undergoes programmed cell death (apoptosis) at the completion of bone resorption.

Unlike the cells that we have seen so far, osteoclasts come from HSCs instead of MSCs [7]. Various factors are important in differentiation and survival of osteoclast precursor including RANKL and CSF-1.

Osteoprotegerins inhibit their actions on RANK receptor. The degree of osteoclast development and function is determined by the RANKL/OPG expression ratio. Osteoclast development depends on signaling through mononuclear precursor cell colony stimulating factor receptor. This signaling then upregulates the secretion of RANK and regulates the key osteoclast gene. These genes are required for both the smooth functioning of mature multinucleated osteoclasts and the maturation of osteoclast precursors. In inflammatory diseases like rheumatoid arthritis (RA), pro-inflammatory cytokines and RANKL work in conjunction to stimulate osteoclast formation that is further enhanced by the conversion of dendritic cells into osteoclasts.

Osteoclasts which are mature and non-polarized become activated and are attached to the matrix of bone to cause the resorption. Osteoclasts become polarized when they bind to bone. After adhering to the bone matrix, different kinds of podosomes and domains are formed by the polarized osteoclast.

Podosomes have an actin base encircled by a ring complex consisting of some integrins and cytoskeletal proteins while domains contain SZ, RF, and active secretory domains. After the formation of a ruffle border, some vesicles are shifted toward the membrane via tiny tubules. These vesicles contain cathepsin K and metallo-proteinases. At the ruffled border, various enzymes, including cathepsin K, are exocytosed. When the osteoclast’s fibrillar actin cytoskeleton come in contact with the bone matrix, an actin ring develops. This creates SZ that separates the surrounding bone surface from the acidified resorption compartment. Bone resorption becomes impeded if either the RB or the SZ is disrupted.

Osteocytes endocytose the fragments of degraded collagen which release the Ca²⁺ and P which are then released at FSD prior to entering the blood stream. Proteolytic enzymes like cathepsin K additionally break down the collagen fragments during this transcytosis process. Small GTPases regulate every stage in the resorption of bone (Figure 4) [11].

Figure 4.

Osteoclast with their ruffled border and sealing zone and the vesicles that contain H+ and cathepsin moving toward the membrane. This entire arrangement is a sealed area where resorption occurs.

Microscopic analysis of osteoclasts show that a small, specialized surface of the osteoclast is responsible for resorption. The entire arrangement provides a sealed area where resorption occurs. This enclosed region holds lysosomal contents. The pH is around 4, which is acidic and optimum for lysosomal enzyme to break the bone matrix [5].

Several hormones, that is, calcitonin and PTH, and growth factors such as IL-6 regulate osteoclasts. One contributing element to the condition of ‘osteoporosis’ is the last hormone, IL-6. Two molecules that are generated by osteoblasts, osteoprotegerin and RANK ligand, interact to influence osteoclast activity as well. These chemicals additionally regulate osteoclast differentiation (Figure 5) [12].

Figure 5.

A visual overview of bone cells that are responsible for the formation of bone and its metabolism, remodeling, and repair, including osteoblasts, osteoclasts, osteocytes, and osteogenic cells with their significant features.

There are four different kinds of cells in bone tissue. Osteoblasts are formed by undifferentiated osteogenic cells. Osteoblasts deposit in the bone matrix. When osteoblasts get stuck in the calcified matrix, osteocytes form. Osteoclasts are the particular kind of cell lineage that performs the job of bone resorption (Table A1).

1.7 Bone matrix

Bone matrix comprises two components: organic and inorganic. Around 20% of bone mass is made up of organic matrix, mainly composed of collagen. Among them collagen type 1 accounts for 90% of the total bone matrix having less quantity of collagen type III, V, X, and XII (Figure A1).

Collagen is a highly branched protein made up of about 1,000 amino acids, organized into a rope-like structure. Collagen provides pliability to the bone; meanwhile, rigidity of the bone is provided by the inclusion of minerals to collagen. If collagen does not contain additional minerals tissues of bone, it will become extremely pliable and possess qualities comparable to a rubber band.

In addition to collagen, proteoglycans and non-collagenous proteins constitute a minor portion (approximately 10%) of the organic matrix’s bulk. These include mainly BSP and osteopontin [13].

The main inorganic components of bone include calcium and phosphate ions. Bone matrix has complex and well-organized framework that perform a crucial role in maintaining bone’s homeostasis by providing mechanical support. The release of different molecules from the bone matrix may cause the disruption of function of bony cells that leads to bone remodeling [6].

2. The bone remodelling process

Bone undergoes remodeling continuously during the course of an individual’s life. In order to maintain structural integrity and metabolic function, bone remodeling is crucial. The remodeling cycle comprises five coordinated steps that takes place within the basic multicellular unit and occurs at different places across the skeleton, simultaneously yet asynchronously. This process involves both local and systemic regulation through various kinds of hormones and growth factors along with some pathways such as canonical Wnt signaling and RANKL/OPG. In vertebrates, the bone performs a variety of functions, such as providing structural support for muscles, storing and releasing growth hormones stored in the matrix, and protecting vital organs and hematopoietic marrow activity [14]. The bone uses its cellular machinery to modify its structural design and material composition to respond various loads [15]. Processes of “construction” and “reconstruction” that occur in bones throughout the life are “bone modeling” and “bone remodeling.” In modeling, major changes in the bone structure is brought about through a separate process in which bone resorption and formation occur at different sites on a skeleton. However, in remodeling resorption and formation are closely correlated to one another both spatially and temporally, maintaining the same volume and structure of the bone [16].

• A structure called BMU where both bone cells, that is, OB and OC work together during this process; however, its organization differ in different bones. This implies that cortical bone undergoes approximately 2–5% of remodeling per year.

• This structure creates a canal which is cylindrical in shape, 1000 μm in length and 150–200 μm in width in the cortical bone. A circular tunnel is formed by 10 osteoclasts in the dominant loading direction during a cycle, and multiple osteoblasts fill this. This implies that about 2–5% undergoes remodeling every year.

• The remodeling is more active in trabecular bone than cortical bone as it has a significantly greater surface to volume ratio. Osteoclasts dig a trench approximately 40–60 m deep by travelling at a speed of around 25 m/day across the trabecular surface [17].

• Bone modeling and remodeling maximize bone strength while reducing mass to satisfy loading and mobility requirements. Where bone is needed, it is deposited by bone formation; where it is not, it is eliminated by bone resorption. Bone modeling mostly takes place during growth.

• It depends on surface of bone, mostly affecting the three inner surface components of the bone, and it is quite less common on the outer bone surface [15].

• Activation, resorption, reversal, formation, and termination are the five stages of bone remodeling cycle. This cycle takes place over several weeks. Bone-lining cells encircle each BMU, providing a unique setting for coupled resorption-formation. The overall size and volume of the bone remains constant during physiological bone remodeling [11].

2.1 Bone modeling

Modeling initiates in early skeletal development in which bone formation and resorption are non-parallel causing changes in the configuration of bone [16]. This process is in contrast with the remodeling process in which bone cell activity is synchronized and happens on particular bony surfaces, while in modeling activity it is unsynchronized or uncoupled and occurs on different sites [18]. This process is done by various drifts that change bones’ shape by adding or removing bone tissue from an existing surface to withstand mechanical loads [13]. To achieve this, both OB and OC work separately from each other in space. There are two types of modeling: formation and resorption modeling. Both are done by OB and OC, respectively, having a principal objective of changing the shapes of bones and increasing their mass [18].

When these mechanisms are disrupted, as in the case of infantile osteogenesis imperfecta treated with antiresorptive drugs (bisphosphonates), inhibition of this process at metaphysis may occur and exaggerated at diaphysis. In this case, the overall diameter increases with age. Although most bone modeling is finished by when the skeleton reaches adulthood, modeling may still happen in some situations, like during exercise and stress or renal bone disease (Figure 6) [16].

Figure 6.

Bone modelling with the help of osteoblasts and osteoclasts that gives the bone strength to withstand stresses. Yellow color indicates bone formation modeling because of the activity of osteoblast, and grey color represents resorption modeling by the activity of osteoclasts.

2.1.1 Events that signal modeling

Local tissue strain initiates bone modeling. Bone is formed when these stresses are above normal, and formation modeling may occur. Bone is removed, and resorptive modeling begins if stresses are minimal.

The bone cells that detect and respond to mechanical stress are called osteocytes. By the canaliculli, fluid ebbs and flows through compressive forces. Osteocytes sense fluid movement through a structure that protrudes from their cell membrane and is known as the primary cilium. The cilium’s movement incites signaling pathways in the osteocyte, which raise cytosolic calcium levels and alter gene expression.

Osteoclasts and osteoblasts can be stimulated by the products of these genes to start the process of bone molding. A rise in cytosolic calcium in a single osteocyte may spread to other osteocytes because osteocytes are linked by gap junctions. There are two stages of the modeling process: activation and either formation or resorption.

In addition, the stimulation of bone lining cells to differentiate into mature, functional osteoblasts begin forming a matrix. When an adequate quantity of bone mass is added to bear the stress, this process terminates [18].

2.1.2 Bone remodeling

The remodeling process involves bony reconstruction by eliminating discrete, measurable “packets” of bone and replacing them with fresh bone. This happens continuously throughout life, resulting in the growing, adult, and senescent skeletons’ ongoing the remodeling of bone [13].

For sufficient physiological bone remodeling to be achieved, proper coupling of formation and resorption is necessary. This is done via direct communication of various bone cells and occurs in a structure called BMU. This structure plays an important role in facilitating this cycle [19].

The BMU is different in its composition and arrangement among different bones and is surrounded by different cells that resemble a canopy to form an area where OB and OC are anatomically coupled to cause remodeling which is called BRC [16].

The cycle of bone remodeling occurs over several weeks and comprises five steps: first activation then resorption after that reversal, then creation, and finally termination [11].

2.1.3 Activation

This represents the very first stage in bone remodeling. The bone is in a quiet or quiescent condition before this. The bone receives and detects the initiating remodeling signal. This signal may be mechanical or hormonal.

It is of two types: targeted and non-targeted. Remodeling that happens because of mechanical forces and micro-damage is termed as targeted. On the other hand, non-targeted remodeling, which is not site-specific, happens because of systemic alterations in various hormones. Osteocytes are also capable of recognizing the biological signals resulting from physical forces. When the signal is detected or recognized, the BLCs begin to retract, collagenase disintegrates the inner membrane of bone, and precursors of osteoclasts are drawn from the bloodstream and are activated.

After their activation, these began to differentiate and start secreting H⁺ and several enzymes that initiate the resorption of the bony matrix. When serum calcium levels drop, the parathyroid gland secretes PTH to keep normal homeostasis. It affects the kidneys and bones directly and also the intestines indirectly. This hormone promotes RANKL or MSCF expression to promote osteoclast activation and differentiation by attaching at their receptor site on OB and bone’s stromal cells. Additionally, estrogen inhibits the synthesis of RANKL by osteoblasts and osteocytes while concurrently producing OPG from these cells, which decreases the formation of osteoclasts. Thus, as estrogen levels drop among women after menopause, osteoclast survival and production rises, leading to an increase in bone resorption [20, 21, 22, 23].

2.1.4 Resorption

During each remodeling cycle, osteoclast-mediated bone resorption lasts approximately about two to four weeks. The cytoskeleton of osteoclasts is reorganized, leading to adhesion to the surface of the bone. Bone OC cells pump proton H⁺ in the compartment which is formed by the creation of SZ and RF to increase the area where this secretory activity takes place. This results in the destruction of bone minerals.

Various other enzymes are also released to cleave minerals of bone, and this includes metalloprotease or cat K etc. This leads to the formation of cavities on the surface of trabecular bone that are referred to as Howship’s lacunae. The resorption phase is then terminated by the multinucleated osteoclasts undergoing apoptosis [11, 13, 16, 24].

2.1.5 Reversal

We still are not entirely familiar with the reversal phase, which is when bone resorption transforms into formation and lasts approximately four to five weeks. Two major events are believed to be taking place, though. First, the new bone matrix is deposited onto the freshly resorbed bone surface, and further signaling takes place to couple resorption to the formation and prevent net bone loss.

Several coupling signals have been postulated, including the substances generated from the bone matrix, that is, TGF-β whose quantity is equated with markers of turnover in the matrix of bone-like type 1 collagen pro-peptide as well as serum OC. TGF-β reduces osteoclastic resorption by inhibiting osteoblasts from producing RANKL.

Theories suggest that, with their surface regulating receptor and cytokines, osteoclast is the primary source of the factors that are responsible for coupling. Some other factors also included in this are IGF BMP-2 and TGF-b [11, 13, 16, 24].

2.1.6 Formation

The process of bone formation might take four to six months. Osteoblasts form the new, proteinaceous matrix to fill up the cavities that osteoclasts left behind [11].

Several potential coupling mechanisms have been suggested, such as the cell-anchored EphB4ephrin-B2 bidirectional signaling complex and the soluble chemical sphingosine 1-phosphate. Lyso-sphingolipids (S1P) are released by OC which prompts OB precursors’ enlistment and increases their survival. Osteoblasts demonstrate EphB4 receptors, while osteoclasts express the ligand Ephrin-B2 that increases osteogenic differentiation by forward signaling.

Both processes of stimulating creation and inhibiting the resorption of bone are achieved by this signaling (eph/ephrin). As a result, coupling may need to occur through a variety of different processes, including soluble signals and direct contact. When osteocytes are at rest, they inhibit bone growth by terminating the Wnt signaling via sclerostin, but when stress is applied, they activate PTH signaling which then promotes bone growth by inhibiting their sclerostin action and maintaining BMD of bone but is still unclear how PTH signaling and mechanical strain interact to enhance remodeling.

MSCs may undergo differentiation to form new bone when MSCs along with OB progenitor cells move toward resorption lacunae. Type 1 collagen is the primary organic substance which makes up bone along with other organic substances and protein as discussed earlier; however, this freshly deposited osteoid is amalgamated by hydroxylapatite [25]

The complex procedure of bone mineralization, that includes the deposition of hydroxyapatite crystals among collagen fibrils, is poorly understood. The ratio of PPi to P is the major regulator in bone mineralization which is affected by a number of factors. Here, PPi is called inorganic pyro-phosphate, and P represents phosphate. Between 50% and 70% of osteoblasts undergo apoptosis after the completion of bone formation, and the remaining osteoblasts convert into osteocytes, or bone-lining cells [11, 13, 16, 20].

2.1.7 Termination

The final stage, which initiates after one month of formation of osteoids, is known as mineralization or calcification and ends after 3 months in trabecular and 120–130 days in cortical bone after the formation of this osteoid. The remodeling cycle is complete when a comparable volume of bone is regained that is being resorbed (Figure 7) [11, 16, 25].

Figure 7.

Bone remodeling compartment: the adjacent bone lining cells receive signals from osteocytes through the canalicular network when bone remodeling is required. After that, a compartment is created where this process occurs by shrinking of these cells called as BRC [24].

The bone remodeling cycle begins when pre-OC comes in contact with bone marrow capillaries and differentiate into mature osteoclasts under the action of pro-osteoclastogenic cytokines derived from osteocytes, such as M-CSF and RANKL. Pre-osteoclasts are pulled in this compartment BRC in which they are converted into OB that forms bone and load up lacuna. This process is believed to be induced by signals derived from osteocytes.

3. Endocrine regulation of bone remodeling

3.1 Parathyroid hormone (PTH)

Parathyroid glands release a polypeptide hormone from their main cells that serves to raise blood calcium levels. PTH affects the kidney and bone directly, and it also indirectly affects the intestines by the action of vitamin D. Furthermore, PTH regulates bone mass in an endocrine way [17, 19, 26].

Depending upon the length of exposure, PTH might have a directly opposing impact on remodeling. Loss of bone mass in both cortical and trabecular bone is because of continuous PTH, whereas cortical bone loss is more severe. The alterations in the OPG-RANKL-RANK signaling pathway by PTH are responsible for these catabolic consequences. Osteocytes and osteoblasts are the source of continuous PTH, which stimulates osteoclastogenesis by inhibiting OPG and increasing RANKL [26].

Lower plasma calcium concentrations activate the negative feedback process and cause less binding to the parathyroid gland’s calcium-sensing receptors (CaSR). As a result, more PTH will be released, increasing the levels of calcium. This hormone also affects OC indirectly by increasing RANKL activity which controls their action and results in more plasma calcium levels.

On the other hand, binding to the CASR receptor is lessened when the plasma calcium concentration is increased and this will inhibit PTH release. When CaSRs are stimulated, the receptor undergoes a conformational shift that stimulates the phospholipase C pathway. This eventually results in increased intracellular calcium that inhibits PTH from being exocytosed from the parathyroid gland’s chief cells.

On the other hand, to treat osteoporosis, PTH is administered intermittently and acts as an anabolic agent. Sclerostin along with dickkopf-1 are expressed less when PTH signaling is intermittent, whereas Wnt ligand Wnt10b is expressed more. Osteoblastogenesis and bone formation increase when canonical Wnt signaling increased [16, 26].

3.2 Estrogen

Estrogen is the primary hormone that regulates bone metabolism in both men and women [27, 28, 29].

Estrogens promote the formation of new bone while inhibiting resorption. Men with insufficient levels reconstruct at a faster rate because they produce less estrogen from testosterone by aromatization [16].

In cases of estrogen insufficiency, there is an increase in bone remodeling which causes a reduction in bony mass because resorption is much more than formation [26].

The concept that osteoporosis results from decreased bone formation after menopause was initially proposed by Albright et al. in the 1940s. IL-1 and TNF production suppression is done by estrogen. These are not only well-known inhibitors of formation but also a powerful bone resorption promotor.

They directly affect osteoblast cells, which in turn indirectly activates adult osteoclasts. According to certain studies, TNF and IL-1 stimulate osteoclast precursor activity directly, which increases the production of osteoclast cells. The action of these cytokines is blocked by estrogen. Estrogen indirectly limits IL-6 expression. The actions of TNF and IL-1 that upregulate IL-6 and assist its actions are blocked, and that is why, this happens. IL-6 mainly stimulates the process of osteoclastogenesis by stimulating osteoclast precursors. As estrogen inhibits osteoclastogenesis, it therefore has a bone-protective effect. This hormone activates estrogen osteoprotegerin (OPG) and facilitates mature osteoclast’s programmed death directly. This OPG then regulates bony mass [30].

3.3 Calcitonin (CT)

C-cells of the thyroid gland release a hormone called calcitonin in response to elevated calcium levels that inhibit bone resorption by binding to osteoclast’s calcitonin receptor, decreasing their number, secretory activity, and formation of ruffled borders [26, 31].

It was also said that the main regulator of bone resorption process is calcitonin. Because of this, calcitonin has been used widely in clinics to treat bone conditions such osteoporosis, hypercalcemia, and Paget’s disease [32].

It is also believed that osteoblast-like cells lack calcitonin receptors, but in certain systems, this hormone reacts to OBs and enhances the osteoinduction of rhBMP-2. However, the effects of this hormone are still unclear on osteoblasts [19].

3.4 Thyroid hormone

The relationship between thyroid hormones and bone formation was first understood in the 1890s when von Recklinghausen noted a patient with hyperthyroidism and multiple fractures [33].

Thyroid hormones T₃ and T₄ along with TSH cause expansion of epiphyseal plate of long bones via OB stimulation [26].

Thyroid hormone exerts a significant effect on bone and mineral metabolism and is an important modulator of bone remodeling [34, 35].

About 100 years later, we still know that hyperthyroidism causes a 10% loss of bone by increasing their turnover, slowing or delaying their remodeling process, and uncouples osteoblastic and osteoclastic activity. This might lead to osteoporosis. On the other hand, adolescents with hypothyroidism suffer delayed skeletal development and maturation, epiphyseal dysgenesis, and retarded long bone growth. Adult hypothyroidism has been associated with decreased bone turnover and osteosclerosis; both conditions can be resolved frequently with thyroid hormone therapy [16, 29].

Triiodothyronine (T3), a physiologically active thyroxine derivative, influences both osteoblasts and osteoclasts in vitro. T3 affects osteoblasts in two ways: it has been noticed that it inhibits osteoblast proliferation and promotes osteoblast differentiation in both primary and calvarial osteoblasts and osteoblastic cell lines. In osteoclasts, T3 acts directly or indirectly through cytokines and stimulates osteoclast activity [19].

The thyroid hormone receptors (TR) TRα1 and TRβ1 which are encoded by Thra and Thrb gene, respectively, modulate the actions of TH on bone [35].

3.5 Glucocorticoids

Glucocorticoids have a significant impact on the function, differentiation, and replication of bone cells. They promote bone resorption by increasing the expression of collagenase 3. Glucocorticoids have three main effects on osteoblasts: (a) increase their programmed cell death, (b) decrease their differentiation, and (c) reduce their activity by altering the expression of binding proteins and various growth factors. Glucocorticoids increase the risk of osteoporotic fractures by causing rapid bone loss. In cartilage, these hormones inhibit linear growth by modifying GH and IGF [36].

The bone resorption is largely because of direct actions of glucocorticoids on the skeleton and partially due to an increase in calcium excretion in urine and a decrease in an intestinal absorption of calcium. The mechanism by which glucocorticoids oppose the effects of vitamin D in vivo by inhibiting intestinal calcium transport is still unclear [37].

Secondary hyperparathyroidism has been suggested due to elevated renal calcium losses and decreased absorption of calcium, but in glucocorticoids-induced-osteoporosis, it does not seem to be significant.

It is suspected that increased sensitivity to parathyroid hormone (PTH) contributes to the observed bone resorption. As parathyroid hormone/PTH-related peptide’s expression is increased by the glucocorticoids, consistent increase of blood PTH or a pattern of bone loss similar to hyperparathyroidism should be expected if the process results in a hyperparathyroid state that occurs in a glucocorticoid-induced-osteoporosis (GIO). But there is no correlation between the acute or long-term use of glucocorticoids and serum PTH levels in the hyperparathyroidism range. Further evidence from bone densitometry suggests that PTH is not involved in glucocorticoid-induced osteoporosis.

A condition that is recognized by cortical bone loss while cancellous bone is preserved is primary hyperparathyroidism; in contrast, glucocorticoid-induced-osteoporosis (GIO) shows an opposite pattern of preferential bone loss. It is confirmed by histomorphometric investigation of bone biopsies that glucocorticoids-induced-osteoporosis and hyperparathyroidism are two different disorders. Osteoblast number is preserved, but bone turnover is increased in primary hyperparathyroidism.

On the other hand, the turnover of bone along with the loss of OBs occurs in GIO. These explanations suggest that PTH do not prominently cause glucocorticoid-induced osteoporosis. When pre-osteoclasts’ RANK receptors are stimulated by the attachment of RANKL, it in association with CSF-1 prompts osteoclastogenesis.

In order to prevent osteoclast receptor from attaching the RANKL, osteoprotegerin binds to RANKL and functions as a decoy receptor. RANKL and CSF1 expressions are upregulated by glucocorticoids, but osteoprotegerin expression is downregulated in the surrounding stromal cells and osteoblasts. Consequently, there is an increase in the production of osteoclasts and bone resorption [26, 36].

When exposed to glucocorticoids, bone resorption is inhibited by bisphosphonate, which also stops and reverses bone loss. But evidence from clinical setting states that people with GIO have bone loss in the starting months when they are exposed to glucocorticoids. Anabolic substances can enhance bone mass in the GIO by promoting bone formation, such as parathyroid hormone [36, 37, 38].

3.6 Growth hormone and IGF

Growth hormone is released from anterior pituitary gland under hypothalamic regulation. It plays multiple roles in the human body, including control of several metabolic pathways, regulating the release and the function of other hormones, and interacting with the immune system. But as its name suggests, the most studied task it performs is coordinating longitudinal growth. Through the local and systemic synthesis of IGF-I, GH affects directly as well as indirectly. As the development and metabolism of bone is controlled via both GH and IGF-I, they also influence bone mass. Throughout childhood, bone mass gradually grows, reaching a peak in the middle of 20s. There is a consequent gradual decrease that quickens at later age.

In childhood, both growth and remodeling causes bone mass development. In order to promote bone formation, GH both directly and indirectly increases osteoblast proliferation and activity through IGF-I. It has been found that osteoblastic MC3T3-E1 cells have GH binding sites [39].

In addition to that, it also increases osteoclast activity and differentiation which promotes bone resorption. As a result, there is a net increase in bone accumulation and raise in the overall momentum of the remodeling process. When GH lacks, bone remodeling occurs more slowly and eventually loses bone mineral density. Although GH directly affects chondrocytes, it mostly controls their function by stimulating the formation of matrix and cell proliferation in these cells through IGF-I [26, 40, 41].

A long-term GH therapy may reverse the severe limitation on bone growth and a decrease in bone mineral density caused by GH deficiency [42].

4. Bone growth or ossification

A process that begins at 6–7th week of embryogenesis and goes till the age of 25, in which bone formation occurs, is known as ossification or osteogenesis of bone. This process varies in different individuals and in different bones [43].

There are two types of bone ossification: intramembranous and endochondral ossification. In both cases, pre-existing mesenchymal tissue is converted into bone tissue. Intramembranous ossification refers to the process of directly converting mesenchymal tissue into bone to form flat bones. This procedure primarily occurs in the skull’s bones. On the other hand, the mesenchymal cells may develop into cartilage, which is then converted to bone [44].

4.1 Intramembranous ossification

This process does not begin with an existing cartilage model. Intramembranous ossification is the process through which most of the skull’s bones and a few other (flat) bones, including the clavicle and scapula, forms embryonically. Intramembranous ossification begins in mesenchyme, which is embryonic connective tissue having mesenchymal cells. This typically is linked to embryonic development, and intramembranous ossification may also occur after birth (during bone repair or healing). During their first stage, a blastema is formed which is made by the combination of the mesenchymal cells. This produces bone matrix after their OB differentiation. To direct their cells toward the osteoblastic lineage, RUNX plays a key role.

The process in which formation of bone takes place within a particular area is known as the primary ossification center, which is established when osteoblasts produces the initial bone matrix. Thus, more and more matrix is produced, and few osteoblasts get encapsulated and transform into osteocytes.

Woven bone is formed by initial OBs. More osteoblasts are drawn to the surface, where they continue to produce this bone and then lamellar bone and continue to form the matrix until the required matrix is formed. Some bones, during development, may form as a result of the union of many tiny bony islands. In some bones, including the jaw, that is formed by the intramembranous ossification, there is the creation of some cavities to allow vessels to invade the ossification core (Figure 8) [18, 24]^.

Figure 8.

The intramembranous ossification taking place in the sea of mesenchymal cells. (A) In the beginning, mesenchymal stem cells assemble into blastema and convert into osteoblasts. (B) Production of bone matrix. (C) Bone matrix undergoing remodeling. (D) Photomicrograph showing the jaw’s intramembranous ossification-forming island of bone (eosin and hematoxylin stain) [18, 20].

4.2 Endochondral ossification

A process by which majority of bones, that is, skull base bones (including ethmoid and sphenoid bone), the axial (ribs and vertebrae) and the appendicular bones, long bones, the medial end of the clavicle, as well as short bones are developed is called endochondral ossification [21].

Shrinking of mesenchymal cells, similar to intramembranous ossification, starts this process. These cells differentiate into chondroblasts rather than of osteoblasts. SOX-9 is the transcription factor that drives this process. These cells, called chondroblasts, create a matrix of cartilage that eventually surrounds certain other cells, converting them into chondrocytes. The perichondrium surrounds the hyaline cartilage. Osteoblastic differentiation occurs on this cartilage and initiates building the bone. The transcription factor RUNX2 controls this osteoblast differentiation process, much like it does during intramembranous ossification. The long bone’s diaphysis, or midshaft, is where bone growth first takes place, giving rise to a structure known as the bone collar.

After the formation of this structure, calcification of matrix occurs because the cartilage cells die. Through the osteoclast assistance, there is the entry of primary blood vessel via this collar, in the area of calcified cartilage. These vessels help in the transportation of osteoclasts which not only provide nutrients for remaining cells but also form the primary ossification center [18]. Primary and secondary sites are two different locations where this process takes place, and among them, the bone initially grows at the primary site. After that, growth at epiphyseal plate occurs which is responsible for longitudinal growth. This process involves five steps [22].

4.2.1 Zones of endochondral ossification

4.2.1.1 Resting zone

The resting zone is the furthest region from cartilage template’s margins composed of chondrocytes embedded in the hyaline cartilage matrix. At some places, this zone is named as the reverse zone [23].

The chondroblasts adjacent to the perichondrium continually generate new resting zone matrix that is rich in type II collagen. The chondrocytes however are embedded in the matrix to generate a new matrix. Both morphological and physiological properties of the chondrocytes in the resting zone are similar to those of hyaline cartilage in other body parts.

4.2.1.2 Proliferative zone

As the name suggests, the proliferative zone, which is the second area, is the place of active chondrocyte mitosis as shown in Figure 9. The stacked coin appearance of this region is the result of the longitudinal division of cells which makes it readily identifiable histologically. These cells produce significant amounts of type II collagen-rich matrix. Various growth factors, signaling pathways, and proteins regulate the zone of proliferation. Some of these are somatotropin, IHH, IGFs, and BMPs. Among few substances that has been demonstrated to stop chondrocyte proliferation in this region is fibroblast growth factor (FGF).

4.2.1.3 Hypertrophic zone

It is the third zone in which cells are arranged in two portions; among them, lower zone’s cells continue to grow and undergo death, while higher zone’s cells cause long bone growth. Thyroxine along with Wnt-b-catenin pathways is responsible for increase in cartilage cell’s size, while PTHrP and IHH inhibit hypertrophy of cartilage cells. This hypertrophy causes the cell to grow and synthesize matrix that is rich in type 2 collagen. However, this area also exhibits a shift in type II collagen production to type X collagen. Genetic programs lead to a sharp increase in chondrocyte cell size, a shift in production of type 2 toward COL10A1, and the induction of substance like VEGF and ALP that causes the cartilage matrix to calcify and vascularize all components of hypertrophic differentiation [45].

4.2.1.4 Zone of calcification

It is the stage where cartilage calcification is observable. Because of inadequate cellular waste removal or nutrient diffusion, the cells of the cartilage may die in this zone.

4.2.1.5 Zone of ossification

The zone of ossification is the last zone, where bone initially forms. Toward this calcified tissue surface, osteoblasts are drawn from the skeletal tissue to build woven bone.

Osteoblasts that are recruited to the surface of calcified tissue (to form new woven bone) form the skeletal tissue. Osteoclasts also exist in the ossification zone and act to remove calcified cartilage as well as newly formed woven bone, which is through remodeling subsequently converted into lamellar bone (Figure 9) [18].

Figure 9.

Endochondral ossification of bones cells passing through different phases of resting zone, proliferative zone, hypertrophy zone, calcification, and ossification zone to convert the cartilage template into bone to facilitate the growth of bone.

Acknowledgments

We would like to express our gratitude to the authors from the Department of Physical Therapy at Islamia University of Bahawalpur, the Department of Medical Laboratory Technology at Islamia University Bahawalpur, and Quaid-e-Azam Medical College, Bahawalpur, for their contributions to the writing, editing, and analysis of this paper.

Conflict of interest

The authors declare no conflict of interest.

A. Appendix

Cell type	Function	Location
Osteogenic cells	Develop in osteoblast	Deep layers of periosteum and the marrow
Osteoblast	Bone formation	Growing portion of bone including periosteum and endosteum
Osteocytes	Maintain matrix mineral concentration	Entrapped in matrix
Osteoclasts	Bone resorption	Bone surfaces and at sites of old, injured, or unneeded bone

Table A1.

Bone cells along with their functions and location.

Figure A1.

Components that form bone matrix. Inorganic components consititute the most part [9].

B. Osteoblasts

Some of the osteoblasts possess cytoplasmic projections travel in bone matrix direction and finally reached to osteocyte process. At this stage, this OB has two choices: either they are converted into osteocyte or BLCs or undergo programmed cell death. Surprisingly, in the vacuoles of osteoblasts, there are some ovoid bodies having dense bodies, and some TUNEL-positive structures are found. These finding indicate that they are also able to engulf apoptotic material in an addition to skilled phagocytosis during alveolar development of bone [12, 14].

C. Bone lining cells

Recent studies suggest that bone lining cells anchored the hematopoitic stem cells that gives stimulus to them to remain in their undifferentiated state. These cells play a crucial role in changes that are associated with the bone remodeling promoting HSCs differentiation toward osteoclasts and by using matrix metaloproteinases. These cells prepare bone surface by excluding the unmineralized collagen fibril. During their transformation from osteoblasts to osteocytes, some proteins that form phenotype of osteoblast are no longer produced. These include alkaline phosphatases, collagen type 1 osteocalcin, and bone sialoprotein [15].

D. Osteoclasts

Based on electron micrographs, membrane part that is nearest to the mineralized bone surface is highly convoluted and forms ruffled border. A membrane ring called as the sealing zone, seals the resorption area by tightly connecting to the bone and, is present at the outer edge of the ruffled border. And, it is a site where the osteoclast attaches to the surface of bone and where the actual bone resorption occurs. It is also the site where the osteoclast secretes acid (protons H⁺), which acidifies the surrounding environment and other lysosomal enzymes, such as cathepsin K, which breaks down the organic components of the bone matrix.

E. Bone matrix

Collagen is a fibrous highly convoluted protein consisting of thousands of amino acids. These are arranged into a rope-like structure which is 300 nm in length. Its fibrils are made up of one α2 and two α1 polypeptide chains that assemble to form triple helical procollagen molecule inside OBs. These osteoblasts release the pro collagen molecules causing the assembling of each collagen molecule to form collagen fibril which then together forms collagen fiber.

Without collagen, bone becomes brittle just like a chalk. Various processes and abnormalities including aging and genetics can influence its structure, affect structural integrity of bone tissue, and cause weakening of it and become more prone to fracture than normal.

Proteoglycans and non-collagenous proteins form a minor portion of the organic matrix’s bulk, but they are essential for osteoblast differentiation, tissue mineralization, cell adhesion, and bone remodeling [25].

A minute concentration of bicarbonate, sodium, potassium, citrate, magnesium, carbonate, fluorite, zinc, barium, and strontium also constitute inorganic component in addition to calcium and phosphate. Hydroxyapatite crystals, that have a chemical formula of Ca₁₀(PO₄)₆(OH)₂, are formed when calcium and phosphate ions nucleate [14].

F. Bone modeling and remodeling

F.1 Activation

There is active 1,25 vitamin D3 production from inactive precursor and increases calcium absorption by the kidneys because of PTH (Figure A2). This 1,25 VitD3 influences the resorption of bone indirectly and also increases RANKL and MCSF expression by this. Finally, estrogen’s function in the process of bone remodeling is a bone sparing hormone. Both osteoblasts and osteoclasts express estrogen receptors. By causing pre-osteoclast and osteoclast apoptosis, while inhibiting osteoblast and osteocyte apoptosis, and limiting excessive bone resorption, estrogen plays a critical role in controlling the longevity of bone cells [25, 28, 32].

Figure A2.

An Illustration of the various stage of bone remodeling; activation, resorption, reversal, creation and termination in the bone remodeling cycle.

F.2 Ossification

The zone of reserve in which hyaline cartilage acts as a storehouse for chondrocytes may aid in the process of growth. This photomicrograph’s zone of proliferation, in which chondrocytes proliferate quickly, is located at the top divide and become stacked in a longitudinal orientation. The zone of hypertrophy in which chondrocytes enlarge and undergo terminal differentiation, and they subsequently compress the matrix into aligned spicules and secrete type X collagen to stiffen it. The zone of calcified cartilage is where chondrocytes release osteocalcin and matrix vesicles that cause the matrix to start to calcify by crystallizing hydroxyapatite. The zone of ossification is where bone tissue initially appears. Here, osteoblasts form a layer over the calcified cartilage matrix’s spicules and release osteoid (Figure A3), which then develops into woven bone (that further modified into lamellar bone) (Figure A4) [46].

Figures A3.

Various of the endochondral ossification and involved mechanism.

Figures A4.

Remodeling versus modeling: In bone remodeling, the formation of bones and resorption are simultaneously mediated by osteoclasts at the same site. Its main goal are to regenerate the skeleton and recover micor-damage. The periosteal, endocortical, intracortical, and trabecular envelopes all undergoes remodeling. Bone modeling mostlt take placed during skeletal growth.

Abbreviations

MSCs	mesenchymal stem cells
BMPs	bone morphogenic proteins
BMU	basis multicellular unit
HSCs	hematopoietic stem cells
Sz	sealing zone
Rf	ruffled border
FSD	functional secreting domain
CATK	cathepsin k
BLCs	bone lining cells
BRC	bone remodeling compartment