2. Resorption Stage
It is interesting that the process by which osteoclasts dissolve, degrade, and destroy the bone matrix is not referred to as bone degradation, but rather as bone resorption or bone reabsorption. Intuitively, calling it a bone degradation process may seem easier to understand, but the terminology seems intended to emphasize that the process is not merely for breakdown but for absorption and retrieval. The term underlines the active process by which the byproducts such as minerals, generated from bone degradation, are absorbed into the osteoclasts and then transported into the bloodstream or other metabolic pathways — highlighting the notion of recycling and metabolic recovery.
Cellular Rearrangement and Polarization
When the osteoclast precursor cells recruited for bone resorption become stimulated, mature, and activated, they undergo cytoskeletal rearrangement to form a structure optimized for resorption. In this process, osteoclasts exhibit two very distinctive structural changes: the sealing zone formed by an actin ring and the ruffled border — a highly folded and complexly expanded membrane within the sealing zone. A phenomenon of polarization also occurs in which substances required for bone resorption are directed and concentrated toward the ruffled border. This polarization confines bone resorption to specific areas in contact with bone, allowing osteoclasts to efficiently carry out resorption.
Attachment, Sealing, and Formation of the Ruffled Border
To effectively carry out resorption, a mature osteoclast must firmly attach to the target bone matrix. For this, bone lining cells that cover the remodeling area temporarily contract, creating gaps that allow osteoclasts to access the matrix. At the same time, the mineralized bone matrix becomes exposed — a necessary process for osteoclast attachment. This is essential to prevent the acidic substances and degrading enzymes secreted during resorption from affecting surrounding tissues, as the working space must be tightly sealed. Osteoclasts rearrange their cellular structure to achieve firm attachment.
Osteoclasts utilize a cell adhesion protein called αvβ3 integrin to attach to the bone matrix. Osteopontin, vitronectin, and bone sialoprotein (BSP) in the bone matrix contain RGD sequences, which integrins recognize and bind to. RGD stands for the amino acids arginine (Arg), glycine (Gly), and aspartic acid (Asp). Integrins function as the 'hands' by which cells physically adhere to the extracellular matrix (ECM) or other cells, and the αvβ3 integrin strongly grips the Arg-Gly-Asp sequence to form strong adhesion.
Osteoclasts arrange actin filaments in a circular structure called a podosome to form an actin ring that surrounds the attachment site. A sealed resorption space is created within this ring, known as the sealing zone, where bone resorption is localized. Much like a toilet plunger seals to the floor to block water flow, the actin ring adheres tightly to the bone matrix to prevent acidic substances from leaking outside the cell. The space where the bone matrix is actually destroyed and dissolved inside the sealing zone is also called Howship's lacuna.
Within this sealing zone, the cell membrane expands to form a ruffled border. Lysosomes containing various proteolytic enzymes travel via microtubules to this region, fuse with the cell membrane, and form the folded structure. Lysosomes are surrounded by a phospholipid bilayer and contain more than 60 different hydrolytic enzymes in their acidic lumen.[1] Just as soap bubbles merge to increase surface area, lysosomes fuse with the membrane to create a wrinkled form. Through the ruffled border, substances that dissolve the bone matrix are secreted into the resorption space, and conversely, degraded matrix components are absorbed back into the cell and later exported via transcytosis. Transcytosis refers to the membrane-crossing transport process where materials enter the cell on one side in vesicles and exit on the other. The ruffled border is thus the most dynamic and active part of the osteoclast, functioning as the core zone of resorption.
Autophagy is an intracellular cleansing mechanism that uses lysosomes to digest and recycle damaged organelles or unnecessary proteins. Proteins like Atg5, Atg7, Atg4B, and LC3 play crucial roles in this process. Interestingly, when RANKL binds to the RANK receptor on osteoclasts, activated transcription factors such as NF-κB and NFATc1 induce the expression of autophagy-related proteins, which are involved in podosome and ruffled border formation.[2] Rab7, which regulates membrane organelle transport, moves to the ruffled border in an Atg5-dependent manner and helps with membrane fusion and lysosome delivery. LC3 assists in the fusion of enzyme-containing vesicles with the cell membrane. Autophagy also promotes the secretion of bone matrix-degrading enzymes like TRAP, cathepsin K, and MMP9.
Since dissolving bone is a highly energy-consuming task, autophagy also likely helps osteoclasts convert degraded components into intracellular energy sources. Ultimately, this means that autophagy not only cleanses the cell but also supports osteoclast functionality by aiding in structural formation and energy supply essential for bone resorption.
TRAP (Tartrate-Resistant Acid Phosphatase) is also a key enzyme involved in osteoclast migration to resorption sites, functional activation, and mineral dissolution. TRAP is believed to contribute not only to osteoclast migration but also to their proliferation and activation. TRAP-generated reactive oxygen species (ROS) amplify the RANKL signaling pathway, promoting the differentiation and fusion (multinucleation) of osteoclast precursor cells. Additionally, TRAP, as an acidic phosphatase, works with hydrogen ions (H⁺) to create the acidic environment necessary to dissolve mineral components in the bone matrix.
Once the osteoclast has rearranged its cytoskeleton, attached to the resorption area, and concentrated resorptive substances, it is ready to begin bone resorption.
The Bone Degradation Process
The bone matrix is a complex structure combining organic and inorganic components. Effective bone resorption requires both proteolytic enzymes to degrade the primarily type I collagen organic matrix and an acidic environment to dissolve the inorganic components like hydroxyapatite. Acidifying proteins such as large membrane transporters (mainly V-ATPase), ion channels (ClC-7), and various lysosomal proteins play roles in creating the acidic environment. This acidic environment activates proteolytic enzymes and directly contributes to mineral dissolution. The degraded components from bone resorption are absorbed through the ruffled border and exported to the opposite cell membrane — similar to how an excavator digs and moves soil to the other side.
Proteins for Dissolving Inorganic Components
V-ATPase is a large molecular complex that utilizes the energy from ATP hydrolysis to pump protons (H⁺) from the cytoplasm into the resorption lacuna, thereby creating an acidic extracellular environment. However, if V-ATPase continuously pumps the positively charged H⁺ ions to the bone surface, the cell cytoplasm becomes excessively negatively charged while the resorption space becomes overly positive, creating an electrical gradient (a charge imbalance), which in turn generates electrical resistance that interferes with the pumping of H⁺ ions. To neutralize this gradient, a chloride channel called ClC-7 is needed. ClC-7, located on membranes of the ruffled border, lysosomes, and endosomes, acts as an ion-exchange channel that simultaneously swaps Cl⁻ and H⁺ like a revolving door. In the ruffled border, ClC-7 releases two Cl⁻ ions into the resorption space while retrieving one H⁺ back into the cytoplasm, maintaining electroneutrality and enabling sustained H⁺ pumping. This type of transporter, which simultaneously exchanges two ions in opposite directions, is called an antiporter.
Thanks to ClC-7, electrical balance is maintained, allowing V-ATPase to continue operating. For ClC-7 to reach the ruffled border, it must form a complex with OSTM1 (osteopetrosis-associated transmembrane protein 1), which protects and stabilizes ClC-7 from proteolytic degradation. As previously mentioned, the fusion of mononucleated osteoclasts into large multinucleated cells is a critical step in osteoclast maturation, governed by the NFATc1 pathway. OSTM1 also functions as a negative regulator that prevents excessive activation of the NFATc1 pathway, ensuring normal osteoclast maturation and multinucleation.[3]
Proteins for Degrading Organic Matter
Once the inorganic components are dissolved by acidification, the exposed organic matrix is then targeted for degradation. The primary organic component of bone — over 90% — is type I collagen, accompanied by non-collagenous proteins such as osteocalcin, osteonectin, osteopontin, fibronectin, and bone sialoprotein. The key enzyme that degrades these organic components is cathepsin K, a cysteine protease highly expressed specifically in osteoclasts and most active in acidic environments. It is known to be virtually the only enzyme capable of breaking down the triple-helical structure of type I collagen. Additionally, members of the matrix metalloproteinase (MMP) family contribute to organic matrix degradation, with some MMPs also capable of cleaving type I collagen.
3. Reversal Phase: The Transitional Stage from Osteoclasts to Osteoblasts
Once bone resorption is complete and osteoclasts either undergo apoptosis or leave the resorption site, osteoblasts arrive to take over and initiate new bone formation. However, before this occurs, the remodeling site needs to be tidied and the resorbed surface must be cleaned. This phase serves to smoothly connect bone resorption and bone formation and is considered a transitional step between the osteoclast-driven bone resorption and osteoblast-driven bone formation. During this phase, cells known as reversal cells are observed. Their role is to cover the resorbed surface before osteoblasts arrive and to clean the area by removing debris such as degraded matrix remnants and collagen fragments, thereby preparing and creating an environment suitable for osteoblasts to attach and differentiate. The reversal phase seems to last for several weeks. [4]
The origin of reversal cells has not yet been clearly identified, and several hypotheses exist. One hypothesis suggests they originate from the monocyte lineage, such as osteoclasts or macrophages with phagocytic functions. Another suggests they are derived from immature osteoblast precursors, that is, mesenchymal stem cells that have not yet fully matured. As discussed earlier in the section on osteoclast and osteoblast interactions, growth factors like TGF-β and IGF-1 released by osteoclast activity attract mesenchymal stem cells and promote their differentiation into osteoblasts. It is suggested that some of these cells might take on a transitional role as reversal cells before fully differentiating and maturing into osteoblasts.
Observations indicate that over 97% of reversal cells show positive responses to Runx2, a marker of osteoblasts, but negative responses to monocyte markers, which supports the current dominant view that they belong to the osteoblastic lineage. However, there is a growing perspective that these cells may not represent a fixed single lineage, and further research is necessary to clarify their identity.
[References]
[1] Lysosomal biogenesis and function in osteoclasts: a comprehensive review
https://doi.org/10.3389/fcell.2024.1431566
[2] Osteoclasts: New Insights
https://www.nature.com/articles/boneres20133
[3] Ostm1 from Mouse to Human: Insights into Osteoclast Maturation
https://www.mdpi.com/1422-0067/21/16/5600
[4] The reversal phase of the bone-remodeling cycle: cellular prerequisites for coupling resorption and formation
https://pmc.ncbi.nlm.nih.gov/articles/PMC4130129/
