16.0.4 POJA-L7204 LOCOMOTOR SYSTEM: BONE
INTRODUCTION 4: CELL TYPES IN BONE FORMATION
INTRODUCTION 4: CELL TYPES IN BONE FORMATION
Four types of cells are found within bone tissue. Osteogenic cells are undifferentiated and develop into osteoblasts. When osteoblasts get trapped within the calcified matrix, their structure and function changes, and they become osteocytes. Osteoclasts develop from monocytes and macrophages and differ in appearance from other bone cells.
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1. Osteoprogenitor cells Mesenchymal stem cells-derived osteoprogenitor cells (osteoblast precursor cells) differentiate under the influence of the runt-related transcription factor 2 (RUNX2). This protein favours the gene expression characteristic of the phenotype of the osteoblast. Osteoprogenitor cell proliferation are stimulated by IGF-1 and IGF-2 and BMP’s. Also pulsed electromagnetic field stimulation results in stimulation of osteoprogenitor cells followed by bone tissue regeneration (cave healing bone fractures). |
2. Osteoblasts
The osteoblasts are secretory cells that produce the ECM by secreting type I collagen and bone matrix proteins (BMP’s). The transcription factor Runx2 mediates the differentiating effects of BMP’s, especially BMP2 that regulates a.o. bone formation. The BMPs comprise a.o. osteocalcin and osteonectin (Ca2+ binding proteins), bone sialoprotein (multiadhesive glycoproteins (BSP-1), proteoglycans, alkaline phosphatase (ALP). Increasing amounts of capillaries appear around and within the osteoblastic clusters.
A hyaline (eosinophilic in Haematoxylin-eosin stain) ground substance is deposited as osteoid. This so-called osteoid is defined as collagenous fibres masked by a hyaline ground substance just before calcification (mineralisation) starts. Subsequently transformation of osteoid into bone occurs by mineralisation and osteoblasts are trapped in the bone matrix. The osteoblasts regulate the calcification of bone matrix by secretion of small membrane limited ALP-rich matrix vesicles (50-250 nm). In the electron microscope it appears that osteoid consists of coarse collagenous fibrils tending to fuse with each other, mixed with foci of calcification with hydroxyapatite crystals. Gradually the osteoid becomes fully mineralised and small irregularly shaped strands of bone surrounded by osteoblasts appear i.e., the spicules. Subsequently adjoining spicules merge to form trabeculae of the primary spongy bone. Osteoblasts become surrounded by calcified matrix and turn into osteocytes in their lacuna.
The osteoblasts are secretory cells that produce the ECM by secreting type I collagen and bone matrix proteins (BMP’s). The transcription factor Runx2 mediates the differentiating effects of BMP’s, especially BMP2 that regulates a.o. bone formation. The BMPs comprise a.o. osteocalcin and osteonectin (Ca2+ binding proteins), bone sialoprotein (multiadhesive glycoproteins (BSP-1), proteoglycans, alkaline phosphatase (ALP). Increasing amounts of capillaries appear around and within the osteoblastic clusters.
A hyaline (eosinophilic in Haematoxylin-eosin stain) ground substance is deposited as osteoid. This so-called osteoid is defined as collagenous fibres masked by a hyaline ground substance just before calcification (mineralisation) starts. Subsequently transformation of osteoid into bone occurs by mineralisation and osteoblasts are trapped in the bone matrix. The osteoblasts regulate the calcification of bone matrix by secretion of small membrane limited ALP-rich matrix vesicles (50-250 nm). In the electron microscope it appears that osteoid consists of coarse collagenous fibrils tending to fuse with each other, mixed with foci of calcification with hydroxyapatite crystals. Gradually the osteoid becomes fully mineralised and small irregularly shaped strands of bone surrounded by osteoblasts appear i.e., the spicules. Subsequently adjoining spicules merge to form trabeculae of the primary spongy bone. Osteoblasts become surrounded by calcified matrix and turn into osteocytes in their lacuna.
3. Osteocytes
The osteocytes are terminally differentiated osteoblasts embedded in their own produced osteoid matrix. They maintain contact with each other by long cytoplasmic processes in tunnel-shaped canaliculi surrounded by mineralised bone matrix, and with osteoblastic cells in the periosteal side and the endosteal side as well as with the vascularised mesenchyme. Human osteocytes are long-living cells with a lifespan of 10-20 years, with ageing death occurs due to apoptosis, degeneration/necrosis. Metabolically the osteocytes are still active and involved in the process of mechano-transduction. Each osteocyte is provided with a primary cilium that detects the flow of interstitial fluid within the lacuna and is involved in mechanosensation and molecular signalling in bone.
They respond to mechanical forces applied to the bone in that increased mechanical stimuli promotes bone formation. On the other hand, decreased mechanical stimuli (e.g., immobilization) causes bone loss. With increased mechanical stress interstitial fluid moves through the canaliculi and generates a transient streaming potential which in turn opens voltage-gated calcium channels in the osteocytic membranes. This results in the increases of intracellular calcium, ATP, nitric oxide and prostaglandin E2 (PGE2) synthesis that accomplishes an alteration of the expression in fos and cox-2 genes responsible for bone formation. In adult life additional expression of the insulin-like growth factor (IGF-1) gene results in the increased production of IGF-1 which promotes conversion of osteoprogenitor cells into osteoblasts that starts to produce the ECM. In this way those mechanical stressed regions of a bone appear to exhibit the largest deposition of new bone.
The osteocytes are terminally differentiated osteoblasts embedded in their own produced osteoid matrix. They maintain contact with each other by long cytoplasmic processes in tunnel-shaped canaliculi surrounded by mineralised bone matrix, and with osteoblastic cells in the periosteal side and the endosteal side as well as with the vascularised mesenchyme. Human osteocytes are long-living cells with a lifespan of 10-20 years, with ageing death occurs due to apoptosis, degeneration/necrosis. Metabolically the osteocytes are still active and involved in the process of mechano-transduction. Each osteocyte is provided with a primary cilium that detects the flow of interstitial fluid within the lacuna and is involved in mechanosensation and molecular signalling in bone.
They respond to mechanical forces applied to the bone in that increased mechanical stimuli promotes bone formation. On the other hand, decreased mechanical stimuli (e.g., immobilization) causes bone loss. With increased mechanical stress interstitial fluid moves through the canaliculi and generates a transient streaming potential which in turn opens voltage-gated calcium channels in the osteocytic membranes. This results in the increases of intracellular calcium, ATP, nitric oxide and prostaglandin E2 (PGE2) synthesis that accomplishes an alteration of the expression in fos and cox-2 genes responsible for bone formation. In adult life additional expression of the insulin-like growth factor (IGF-1) gene results in the increased production of IGF-1 which promotes conversion of osteoprogenitor cells into osteoblasts that starts to produce the ECM. In this way those mechanical stressed regions of a bone appear to exhibit the largest deposition of new bone.
4. Osteoclasts are not related to osteoblasts?
At sites where removal of bone occurs large, multinucleated cells are present i.e., the osteoclasts. The polynuclear cells with many lysosomes are found in shallow grooves or resorption bays (Howship’s lacuna) in the bone tissue. Lysosomes stained for tartrate-resistant acid phosphatase (TRAP) are used as marker for osteoclast activity and differentiation.
In contrast to older theories nowadays it is clear that osteoclasts are not related to osteoblasts or osteocytes. Differentiation of mononuclear haemopoietic cells from the lineage of granulocyte/macrophage progenitor cells results in osteoclast precursors. These precursors express two transcription factors c.q. c-fos (human proto-oncogene, a homologue of the retroviral oncogene v-fos) and NF-kB [nuclear factor kappa-light-chain-enhancer of activated B-cells (FRANK), a member of the tumour necrosis factor (TNF) receptor superfamily]. The c-fos encodes a 62 kDa protein that forms heterodimer with transcription factors resulting in the formation of Activator Protein-1 (AP-1) complex which binds DNA at AP-1 specific sites. NF-kB is a protein complex that controls transcription of DNA, cytokine production and cell survival).
At sites where removal of bone occurs large, multinucleated cells are present i.e., the osteoclasts. The polynuclear cells with many lysosomes are found in shallow grooves or resorption bays (Howship’s lacuna) in the bone tissue. Lysosomes stained for tartrate-resistant acid phosphatase (TRAP) are used as marker for osteoclast activity and differentiation.
In contrast to older theories nowadays it is clear that osteoclasts are not related to osteoblasts or osteocytes. Differentiation of mononuclear haemopoietic cells from the lineage of granulocyte/macrophage progenitor cells results in osteoclast precursors. These precursors express two transcription factors c.q. c-fos (human proto-oncogene, a homologue of the retroviral oncogene v-fos) and NF-kB [nuclear factor kappa-light-chain-enhancer of activated B-cells (FRANK), a member of the tumour necrosis factor (TNF) receptor superfamily]. The c-fos encodes a 62 kDa protein that forms heterodimer with transcription factors resulting in the formation of Activator Protein-1 (AP-1) complex which binds DNA at AP-1 specific sites. NF-kB is a protein complex that controls transcription of DNA, cytokine production and cell survival).
5. Effects of cytokines/TNF.
Under the influence of the multiple cytokines and RANK these precursors fuse with another and become multinucleated. RANK appears present on osteoclast precursor cells but also on mature osteoclasts. Osteoblastic stromal cells produce the ligand RANKL and regulation of its mRNA has been shown to correlate with activation of osteoclast genesis. FRANKL has the ability to stimulate the formation of osteoclasts from precursor cells as well as the bone-resorbing activity of mature osteoclasts. During inflammation activated T-lymphocytes can produce both membrane-bound and soluble RANKL molecules. In this way inflammatory processes can stimulate osteoclast-mediated bone resorption. However osteoprotegerin (OPG) appears a potent inhibitor of osteoclast formation. OPG is a member of the cytokine secreted TNF receptor superfamily. In this way OPG, mainly produced by osteoblasts can block that resorption pathway. Under the influence of regulators of bone metabolism, e.g., IL-1, TGF-b, vit B etc., OPG serves as a entrap receptor (or ‘decoy’ receptor) for RANKL.
Under the influence of the multiple cytokines and RANK these precursors fuse with another and become multinucleated. RANK appears present on osteoclast precursor cells but also on mature osteoclasts. Osteoblastic stromal cells produce the ligand RANKL and regulation of its mRNA has been shown to correlate with activation of osteoclast genesis. FRANKL has the ability to stimulate the formation of osteoclasts from precursor cells as well as the bone-resorbing activity of mature osteoclasts. During inflammation activated T-lymphocytes can produce both membrane-bound and soluble RANKL molecules. In this way inflammatory processes can stimulate osteoclast-mediated bone resorption. However osteoprotegerin (OPG) appears a potent inhibitor of osteoclast formation. OPG is a member of the cytokine secreted TNF receptor superfamily. In this way OPG, mainly produced by osteoblasts can block that resorption pathway. Under the influence of regulators of bone metabolism, e.g., IL-1, TGF-b, vit B etc., OPG serves as a entrap receptor (or ‘decoy’ receptor) for RANKL.
6. Mechanically stressed osteocytes secrete PGE2 that affects lymphocyte function and induces stimulation of the production of RANKL. On the other hand, active osteoblasts, depositing osteoid, produce OPG that inhibits RANKL. In this way it is obvious that there is little or no osteoclastic activity in those regions where osteoblasts are depositing new bone. It becomes clear that bone resorption including bone remodelling by osteoclastic activities is dependent on the influence of the combined OPG/FRANKL system.
7. How do activated osteoclasts look like.
Before newly formed osteoclasts start their bone-resorbing labour, they have to be activated and during that process they become highly polarized. Active osteoclast shows the following specialised regions:
In order to resorb bone effectively osteoclasts attach themselves to the bone surface by specialised actin-rich podosomes. The latter enable the cells to form tight seals with the bone matrix and within these sealed zones cell membranes form deep infoldings the so-called ruffling of the border membranes.
In this way the cell surface area is increased for:
At the site of the clear zone the cell membrane exhibits adhesion molecules like integrin extracellular receptors (vitronectin receptor, type I collagen receptor) responsible for a tight seal between plasma membrane and mineralised bone matrix. Transport vesicles containing degraded bone material that is endocytosed at the ruffled border contain distinct amount of TRAP indicating a role in the breakdown of endocytotic material.
Before newly formed osteoclasts start their bone-resorbing labour, they have to be activated and during that process they become highly polarized. Active osteoclast shows the following specialised regions:
- 1. The direct contact zone of osteoclast with bone tissue: the ruffled border.
- 2. The peripheral part of the osteoclast cytoplasm that borders the area of the ruffled border: the clear zone (sealing zone).
- 3. At the opposite side of the ruffled border the basolateral region of the cytoplasm is involved in the exocytosis of digested material.
In order to resorb bone effectively osteoclasts attach themselves to the bone surface by specialised actin-rich podosomes. The latter enable the cells to form tight seals with the bone matrix and within these sealed zones cell membranes form deep infoldings the so-called ruffling of the border membranes.
In this way the cell surface area is increased for:
- Secretion of e.g., proteolytic enzymes, hydrochloric acid, cathepsin on to the bone surface as well as
- Endocytosis of degradation products and bone debris.
At the site of the clear zone the cell membrane exhibits adhesion molecules like integrin extracellular receptors (vitronectin receptor, type I collagen receptor) responsible for a tight seal between plasma membrane and mineralised bone matrix. Transport vesicles containing degraded bone material that is endocytosed at the ruffled border contain distinct amount of TRAP indicating a role in the breakdown of endocytotic material.
8. Decalcification of bone
Decalcification of the bone matrix starts through acidification of the bony surface. The osteoclast contains carbonic anhydrase I that produce carbonic acid, that dissociates into bicarbonate and a proton (H+). Using ATP-dependent proton pumps, these protons are transported through the ruffled border resulting in a low pH in the resorption groove. Excess bicarbonate is eliminated by passive exchange with chloride ions via chloride-carbonate protein exchangers located at the basolateral membranes. In the acidic microenvironment the degradation of hydroxyapatites progresses into calcium ions, soluble inorganic phosphates and water.
The next step after acidification of the microenvironment of the resorption groove is the degradation of organic components (collagen, proteins) of the bone matrix by the secreted hydrolytic enzymes such as cathepsin and matrix metalloproteinases.
Most vesicles in the osteoclast are lysosomes, their content appear to be released in the clefts between the osteoclastic processes of the ruffled border. This is a specific example of extracellular action of released lysosomal enzymes that functions outside the osteoclast.
After the resorption of bone tissue is achieved the large osteoclasts go through the process of apoptosis.
Nowadays it is known that healthy as well as dying osteocytes communicate with osteoclasts in order to engage them for remodelling of bone tissue. At sites of bone damage apoptotic osteocytes produce apoptotic dodies that re-express RANKL molecules. In this way these molecules increase osteoclastic activity via the RANK-RANKL signalling pathways.
8. Decalcification of bone
Decalcification of the bone matrix starts through acidification of the bony surface. The osteoclast contains carbonic anhydrase I that produce carbonic acid, that dissociates into bicarbonate and a proton (H+). Using ATP-dependent proton pumps, these protons are transported through the ruffled border resulting in a low pH in the resorption groove. Excess bicarbonate is eliminated by passive exchange with chloride ions via chloride-carbonate protein exchangers located at the basolateral membranes. In the acidic microenvironment the degradation of hydroxyapatites progresses into calcium ions, soluble inorganic phosphates and water.
The next step after acidification of the microenvironment of the resorption groove is the degradation of organic components (collagen, proteins) of the bone matrix by the secreted hydrolytic enzymes such as cathepsin and matrix metalloproteinases.
Most vesicles in the osteoclast are lysosomes, their content appear to be released in the clefts between the osteoclastic processes of the ruffled border. This is a specific example of extracellular action of released lysosomal enzymes that functions outside the osteoclast.
After the resorption of bone tissue is achieved the large osteoclasts go through the process of apoptosis.
Nowadays it is known that healthy as well as dying osteocytes communicate with osteoclasts in order to engage them for remodelling of bone tissue. At sites of bone damage apoptotic osteocytes produce apoptotic dodies that re-express RANKL molecules. In this way these molecules increase osteoclastic activity via the RANK-RANKL signalling pathways.
9. Hormones affect the balance between bone production and resorption.
The chief cells (or principal cells) of the parathyroid gland produce the parathyroid hormone (PTH). The hormone regulates calcium and phosphate levels in the extracellular fluid. The osteoclasts lack PTH receptors, so the effect of PTH concerning the osteoclasts appears to be indirectly. It is known that T-lymphocytes, osteocytes and osteoblasts expose PTH receptors that activate adenyl cyclase and increase cAMP. Exposure to PTH increases bone mass through the cAMP/IGF pathway in osteoblasts and osteocytes. Prolonged exposure to PTH increases the RANKL production by T-lymphocytes and by osteoblasts. Subsequently this results in hyperactivity of the osteoclasts that may results in osteoporosis. On the other hand, oestrogen inhibits the lymphocytic production of RANKL that contributes to osteoclastic apoptosis.
Nonetheless the parafollicular cells in the thyroid gland produce calcitonin that effectively reduces osteoclastic activity.
The chief cells (or principal cells) of the parathyroid gland produce the parathyroid hormone (PTH). The hormone regulates calcium and phosphate levels in the extracellular fluid. The osteoclasts lack PTH receptors, so the effect of PTH concerning the osteoclasts appears to be indirectly. It is known that T-lymphocytes, osteocytes and osteoblasts expose PTH receptors that activate adenyl cyclase and increase cAMP. Exposure to PTH increases bone mass through the cAMP/IGF pathway in osteoblasts and osteocytes. Prolonged exposure to PTH increases the RANKL production by T-lymphocytes and by osteoblasts. Subsequently this results in hyperactivity of the osteoclasts that may results in osteoporosis. On the other hand, oestrogen inhibits the lymphocytic production of RANKL that contributes to osteoclastic apoptosis.
Nonetheless the parafollicular cells in the thyroid gland produce calcitonin that effectively reduces osteoclastic activity.
10. Verdict
Nowadays it is clear that osteoclasts are not merely bone resorbing cells, they also regulate osteoblast functions and mediate the exodus of hematopoietic stem cells from the marrow into the blood. In pathologic circumstances osteoclasts may act as immunomodulators.
Nowadays it is clear that osteoclasts are not merely bone resorbing cells, they also regulate osteoblast functions and mediate the exodus of hematopoietic stem cells from the marrow into the blood. In pathologic circumstances osteoclasts may act as immunomodulators.