4. Bone Formation Phase
Bone formation is broadly divided into two stages: the formation of an organic matrix centered on collagen, and the subsequent mineralization stage, in which inorganic components (primarily hydroxyapatite crystals) are deposited within that matrix. Here, the term "bone matrix" refers to the extracellular matrix (ECM) excluding bone cells. As seen earlier in connective tissue including joints, a characteristic of connective tissue is a relatively small number of cells and a large proportion of extracellular matrix. Thus, bone formation essentially involves the process by which bone cells produce and assemble the extracellular matrix. The bone matrix is a complex product composed of both organic and inorganic components.
To initiate bone formation, osteoblasts first secrete a protein mixture called the osteoid. This osteoid serves as the basic framework of the organic matrix and consists of more than 90% collagen (mostly type I), along with non-collagenous proteins such as osteopontin, osteocalcin, bone sialoprotein, and fibronectin. The osteoid can be likened to the rebar framework prepared before pouring concrete. Just like rebar is arranged in an orderly fashion at a construction site, collagen is arranged in a regular overlapping manner, ensuring flexibility and tensile strength, which allows the bone to withstand pulling forces. Osteoblasts produce this protein-based matrix and secrete it outside the cell to provide the foundation for mineral deposition.
Matrix Vesicle-Mediated Mineralization Hypothesis
If the main organic component of the bone matrix is type I collagen, the main inorganic component is undoubtedly hydroxyapatite. Among the various hypotheses proposed to explain the process of bone mineralization, the most prominent in recent years is the matrix vesicle-mediated mineralization theory. Matrix vesicles are extremely small extracellular vesicles, 30 to 1000 nm in diameter, secreted by osteoblasts into the extracellular matrix. These vesicles are enclosed in a lipid bilayer membrane. The membrane of matrix vesicles is equipped with various transporters, and their interior contains a variety of enzymes that create an environment conducive to forming hydroxyapatite crystals using calcium and phosphate.
In fact, it is fortunate that the materials for mineralization gather inside these vesicles and that hydroxyapatite forms within them. It would be undesirable for mineralization to occur indiscriminately throughout the body, so utilizing vesicles to localize mineralization is a clever and safe strategy.
Matrix vesicles contain calcium ions (Ca²⁺), inorganic phosphate (PO₄³⁻ or Pi), mineralization-promoting enzymes such as PHOSPHO1 and ALP, and calcium channel proteins like annexins. These factors all aid in the formation of hydroxyapatite crystals. Calcium and phosphate from the extracellular matrix enter the vesicles through membrane channels, gradually binding together inside the vesicle to form needle-like crystals. As more crystals grow and expand, they eventually form spherical nodules that burst through the vesicle membrane and emerge outside.
Even after rupturing, the enzymes and membrane channels within the vesicle remain functional, allowing mineralization to continue. To prevent excessive mineralization, inhibitory factors such as pyrophosphate (PPi), composed of two phosphate molecules, and the protein osteopontin play a regulatory role.
Materials for Hydroxyapatite Formation
With respect to matrix vesicles, we can examine the membrane transporters that allow material inflow and outflow, and the internal enzymes. Since the role of matrix vesicles is to accumulate Ca²⁺ and PO₄³⁻ to produce hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), it is sufficient to transport the readily available extracellular calcium ions into the vesicles. This transport is achieved via Ca²⁺ ATPases that actively pump calcium into the vesicle using ATP, and annexin channel transporters (mainly Annexin II, V, and VI).
In contrast, phosphate is not readily available in usable form extracellularly; it exists mostly as organic phosphate derivatives (ATP, ADP, AMP, NTP, etc.), which must be enzymatically broken down into inorganic phosphate before entering the vesicle.
Enzymes Generating Inorganic Phosphate
Enzymes responsible for generating inorganic phosphate include alkaline phosphatase (ALP), tissue nonspecific alkaline phosphatase (TNAP), ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and PHOSPHO1 (phosphatase, orphan 1). ALP removes phosphate groups from organic phosphate compounds like ATP or ADP to produce inorganic phosphate. TNAP, a subtype of ALP, breaks down pyrophosphate (PPi) generated by ENPP1 into two inorganic phosphates. ENPP1 breaks ATP (containing three phosphates) into AMP and PPi. Interestingly, PPi is one of the inhibitors of excessive mineralization. Therefore, to promote mineralization, PPi must be removed, and TNAP plays this role by converting PPi into two phosphate ions, thus facilitating mineralization. As shown in the figure below, ENPP1 and TNAP function as a sequential enzyme system.
Pyrophosphatase directly breaks down PPi within the matrix vesicle. While most of these enzymes are located on the vesicle membrane, PHOSPHO1 functions within the lipid bilayer to break down phosphocholine and phosphoethanolamine, generating inorganic phosphate and playing a critical role in initiating the nucleation of hydroxyapatite crystals within the vesicle. Pit1 (SLC20A1) and Pit-2 (SLC20A2) are sodium-dependent phosphate transporters that co-transport Na⁺ and phosphate into the vesicle.
Mineralization Inhibitors
Pyrophosphate (PPi) and osteopontin are major mineralization inhibitors that prevent excessive mineralization. The ANK (ankylosis protein) transmembrane channel helps regulate extracellular PPi levels by allowing PPi generated inside matrix vesicles to exit through the membrane.
Nucleation of Hydroxyapatite Crystals
Just as snowflakes form around dust particles in clouds, a nucleation site is required for the formation of hydroxyapatite crystals. This nucleation site is formed inside the matrix vesicle. Since the vesicle is surrounded by a lipid bilayer, the membrane phospholipids, such as phosphatidylserine and phosphatidylinositol, which are negatively charged, strongly attract the divalent Ca²⁺ ions, leading to calcium concentration near the inner membrane surface. Phosphates transported into the vesicle via membrane transporters combine with calcium to form calcium-phosphate-phospholipid complexes, serving as nucleation sites for hydroxyapatite. When the concentration of calcium and phosphate reaches a critical threshold, crystals begin to form, initially in an amorphous state, then transition into needle-shaped crystals and eventually mature into hydroxyapatite crystals. This process is finely regulated by the enzymes within the vesicle to ensure balanced and non-excessive crystallization.
Mineralization of Collagen
Once hydroxyapatite crystals emerge from the vesicle membrane, they form rounded mineralized nodules composed of numerous needle-shaped crystals. These nodules come into contact with collagen fibers abundantly present in the organic matrix (osteoid), initiating collagen mineralization. Osteoblasts continuously secrete matrix vesicles toward the protein-rich organic matrix, and the hydroxyapatite crystals produced from these vesicles infiltrate the spaces between collagen fibers, gradually mineralizing them. At this stage, osteopontin acts to prevent excessive mineralization around the hydroxyapatite nodules.
There are two main hypotheses explaining how hydroxyapatite binds to collagen. The first is the hole zone theory, which suggests that type I collagen molecules, each composed of three intertwined polypeptide chains forming a triple helix, are staggered to form fibrils. This staggered arrangement creates overlapping and gap regions, and the gap zones are where mineral deposition occurs to form hydroxyapatite crystals. The second is the spiral tracking theory, which suggests that minerals are deposited along the spiral alignment of parallel collagen fibers at specific intervals.[1] Both theories agree that hydroxyapatite crystals align along collagen fibers to ensure mechanical strength and structural stability of the bone.
For details on collagen biosynthesis, refer to the next article. The figure below shows how collagen molecules form microfibrils through cross-linking. About a quarter(1/4) of collagen molecules are staggered, and the densely overlapped regions appear dark under electron microscopy due to high electron density (overlap zones), while the less overlapped gap zones appear lighter. The periodic repetition of overlap and gap zones gives collagen fibers a striped appearance. A single D-period, consisting of one 27 nm overlap zone and one 40 nm gap zone, measures 67 nm in humans. Matrix vesicles fit into these gaps, forming hydroxyapatite crystals and that is what hole zone theory suggests.
As these crystals grow and fill the internal spaces between collagen fibers, they expand outward, merging with nearby hydroxyapatite crystals, increasing both size and number. As the crystals diffuse throughout the collagen matrix, the collagen fibers are transformed into rigid, mineralized bone tissue. This crystallization process is tightly regulated by proteins like osteocalcin and osteonectin, which control crystal orientation and size. Once the collagen matrix is densely filled with hydroxyapatite, the bone tissue becomes a completed mineralized structure with high mechanical strength and appropriate flexibility. Mineralization progresses over several months after crystallization begins, eventually maturing into robust bone tissue.
Duration of Bone Remodeling Phases
The time required from the recruitment of osteoclasts to the completion of stable mineralization varies depending on several factors, including age, health status, and hormones (e.g., hyperparathyroidism). Generally, bone resorption lasts about 3 to 40 days, while bone formation takes approximately 120 to 150 days. Cancellous (spongy) bone undergoes more active remodeling due to its high metabolic activity, large surface area, and rich blood supply, but mineralization may proceed more slowly compared to compact (cortical) bone due to its looser structure.
5. Resting Phase: The new fate of osteoblasts
Other osteoblasts become embedded within the bone matrix they have secreted and remain there, differentiating and maturing into osteocytes. These osteocytes reside in small spaces between mineralized collagen fibers called lacunae. Within these lacunae—literally meaning “empty space”—they extend long, slender dendritic processes outward from the main cell body in all directions, giving them a characteristic star-like shape that distinguishes them from osteoblasts.
Osteocytes are directly connected to neighboring osteocytes, bone lining cells, and osteoblasts through microscopic canals known as canaliculi. Through this extensive network, they can communicate and exchange nutrients and waste products. This intercellular network of osteocytes forms a vast web throughout the bone matrix, playing a crucial role in detecting mechanical stress or microcracks in the bone. When such signals are received, the network initiates a prompt remodeling response, making osteocytes essential as sentinels, sensors, and monitors within the bone matrix.
The final phase of bone remodeling is the resting phase. During this period, the bone surface stabilizes and is maintained in a structurally complete state. Similar to animals hibernating during winter, cellular metabolism slows dramatically, and visible cellular activity is minimal. However, the bone remains in a prepared state, ready to reactivate remodeling when needed. This is a resting phase in name, but one full of latent potential.
The overall process of bone remodeling—from the resting phase to bone resorption, new bone formation, mineralization, and the return to resting phase—can be visualized in the diagram below.
[References]
[1] Ultrastructural and biochemical aspects of matrix vesicle-mediated mineralization
https://doi.org/10.1016/j.jdsr.2016.09.002
