Physiology of Bone

The Adult human body is composed of around 213 bones, these provide the body with structure, support, mobility and protection. The composition of bone includes an organic matrix and and and inorganic matrix. The organic matrix is composed of 90% collagenous protein, with this primarily being type 1, this provides the bone with flexibility and tensile strength. Proteoglycans which inhibit mineralisation and enhance the compressive strength. Matrix proteins which promote mineralisation and bone formation. Cytokines and Growth factors responsible for bone cell differentiation, activation and growth.

The inorganic material includes 70% hydroxyapatite, responsible for the rigidity and hardness of bone, as well as small amounts of salts such as magnesium, sodium and bicarbonate.

During our lifespan bones may undergo a series of growth, modelling and remodelling phases both longitudinally and radially.  Growth predominantly occurs from childhood to adolescence, when after this some bones may undergo mineralisation and fuse. Radial growth and remodelling however continues as we age, with the overall shape of bones influenced by physiological forces that they may be exposed to. Often in ageing bones may be seen to widen as they undergo remodelling. Remodelling is essential for the upkeep and and maintenance of bone strength, replacing old bone with newly synthesised protein matrix, which can undergo mineralisation to form new bone. This is a continual process regulated by Osteoclasts and Osteoblasts with 4 sequential phases, activation, resorption, reversal and formation. 

Osteoclasts are produced by hematopoietic stem cells in the bone marrow. They reabsorb bone, with their formation and activation regulated by a number of factors including RANKL, IL-6, OPG, PTH, Calcitonin and 1,25-dihydroxyvitamin D. Mediators secreted in response to inflammation and by macrophages also enhance osteoclast differentiation. Osteoclasts work by secreting H+ ions onto the bone resorption site, lowering the pH. Hydroxyapatite breaks down in this new acidic environment causing the release of calcium ions. Further movement of the bone mineral leads to its digestion by enzymes released by osteoclasts, such as Tartrate-resistant acid phosphatase, cathepsin K, matrix metalloproteinase 9, and gelatinase.

Osteoblasts are produced by stromal marrow cells and form new bone by synthesising collagenous organic matrix. The matrix will gradually be mineralised to produce new bone with osteoblasts supporting this process by releasing vesicles of calcium and phosphate. These inhibit pyrophosphate and proteoglycans known to halt mineralisation. Osteoblasts that move into the bone cavity can differentiate into a new type of cell called osteocytes. Osteocytes lie within mineralised bone where they will extend branches to connect to other cells. They are connected both metabolically and electrically via gap junctions, mainly composed of connexin 43, these connections are vital for the cells survival, activity and maturation. This network can sense and respond to pressures or weakness in the bone from external forces, attracting osteoclasts to the site of repair. Osteocytes also secrete bone matrix proteins that support intracellular adhesion and the movement of minerals. Sclerostin is also released which inhibits further bone formation in nearby osteoblasts. Apoptosis of these cells has been influenced by reduced oestrogen levels which may affect the gap junctions and bone matrix interactions. 

When the osteoblasts have finished filing the bone cavity they will lie flat where they are known as lining cells, these cells can respond to the movement of calcium and other minerals in and out of the bone extracellular fluid. They also remain sensitive to hormones such as PTH that can re differentiate into osteoblasts. Around 50-70% of activated osteoblasts will undergo apoptosis once osteocytes and lining cells have been formed.

The 3 main matrix proteins within bone physiology include osteocalcin, osteopontin and osteonectin.

Osteocalcin is the most abundant non collagenous protein found within bone, accounting for 10-20%. It is a small 49 aa protein (5.8 kDa), highly conserved among vertebrate species.  It plays a crucial role in the development of bone with bone structure built upon its presence. The protein is recognised to be produced by mature osteoblasts where it promotes the mineralisation and formation of bone, also attracting osteoclasts to the site of formation. Osteocalcin is recognised to bind to free calcium ion and hydroxyapatite, where it helps to remodel bone, ensuring calcium and phosphate ions precipitate on the bone rather than other surrounding tissue. Osteocalcin moves around the bone structure directing calcium ions in areas needed for growth. Although OC is not essential for bone formation the integrity of bone within knockout mice was found to be compromised, with them more prone to fracture. This suggests OC may work as a shock absorber helping to protect against fractures. Vitamin K is essential for the functioning of OC as it modifies the proteins shape to enable interactions between calcium ions.  Non carboxylated OC may also be detected within the blood where it is understood to enhance insulin sensitivity. OC may stimulate the proliferation of B cells and insulin expression/ secretion. This process can help to improve glucose tolerance and prevent against diabetes.

Osteonectin, is a 32 kDa protein secreted by platelets, endothelial cells and osteoblasts where it is though to help regulate calcium levels and organise the mineral matrix. Osteonectin can bind to both hydroxyapatite and collagen. As part of the osteonectin collagen complex it can bind to free calcium ions shaping mineral phase deposition and linking minerals to the collagen fibres. The protein is thought to be specific to skeletal tissue with higher levels detected in the matrix rather than the cells  of bones. However its presence in non mineralising tissue also suggests its function is not limited to calcified tissue. From its secretion and expression on platelets and endothelial cells this suggests it may be involved in tissue repair and remodelling, modulating the cell cycle. Where higher levels may be released in response to stress or injury.

Osteopontin is a phosphorylated glycoprotein found within bone, kidney and epithelial linings where is may be secreted into bodily fluids such as milk, blood and urine. Within bone physiology it is thought to regulate bone mineralisation and reduce growth or aggregation of calcium crystals. OPN however may also be linked to other physiological processes such as cell adhesion, migration and survival. It has also been found to be up regulated in inflammation and tissue remodelling, propagating the inflammatory response. OPN levels may also be correlated to diseased states such as Crohns, atherosclerosis, cancer and autoimmune diseases including lupus, MS, rheumatoid arthritis.

A number of hormones are linked to the functioning of bone cells and the release of matrix proteins, particularly PTH and vitamin D.

PTH  (Parathyroid hormone) plays a key role in calcium homeostasis, whereby the hormone increases calcium reabsorption from the kidney tubule and liberation from the bone matrix. PTH is thought to stimulate activity in osteoclasts indirectly as PTH receptors are not displayed on their cell membrane. Instead PTH binds to receptors on osteoblasts which induce the secretion of cytokines, which may then signal the activation of osteoclasts to break down the bone matrix and release calcium ions. PTH is also linked to the increased production and differentiation of osteoblasts. If calcium levels are high within the blood signals are sent to the CaS receptor to reduce the secretion of PTH.

Vitamin D has been found to have a similar role as PTH within the kidney by increasing reabsorption, although the effect is much weaker. Unlike PTH however vitamin D encourages the resorption of phosphate ions which promotes the mineralisation of bone. PTH and vitamin D are found to work alongside one another with PTH signalling to the body to produce more of the active vitamin when calcium levels are low. Vitamin D can be absorbed from foods such as fatty fish or from the sunlight, but the liver and kidney are required to process the vitamin into its active form (1,25(OH)2 D3) following several hydroxylation steps. 

The effects these hormones have on osteocalcin release has been studied however the exact effects are unknown. Studies have indicated that PTH may influence the synthesis of osteocalcin in mature osteoblasts by promoting gene expression. With increased levels of osteocalcin mRNA detected in a number of bone cell lines. The action PTH has on the gene however may be mediated by a number of different signalling pathways. 

In a clinical setting osteocalcin can be used as a marker for bone cell turnover, particularly osteoblast function. High levels of serum OC have been found to correlate well with bone formation rate. However as OC is rapidly degraded in the serum and peptide fragments may be formed, detecting the true level using antibody techniques can be tricky. In addition OC peptide fragments may also be released following bone resorption so it may not be a true representation.  Elevated levels of OC may indicate patients are undergoing increased bone formation often present in diseases such as hyperparathyroidism or Paget’s disease.

Pagets disease is a common bone disorder that affects patients over 50. It is characterised by persistent joint or bone pain, deformities and nerve problems. This is thought to be caused by alterations in the bone remodelling process where osteoclasts are removing bone at a faster rate. The osteoblasts try to counteract this by replacing the  lost bone however this leads to larger weaker bone.  Markers most commonly used to diagnose the disease are serum alkaline phosphatase, bone-specific alkaline phosphatase (BSAP) and Procollagen I N -terminal peptide (PINP).

OC may also be used as a marker for osteoporosis which is a a condition that affects primarily post menopausal women. Bones become weakened, fragile and more susceptible to fractures from the levels of calcium and phosphate decreasing. In addition, as levels of oestrogen decrease this prevents the calcium available from being sufficiently absorbed and utilised for remodelling. Osteoporosis is characterised by a reduction in bone mass density with this found to be directly correlated to increased levels of serum OC. Higher levels of OC are within the circulation as there is less calcium for it to bind to, hindering mineralisation. As OC is tissue specific and shows little variation among the population it can be used as a early diagnosis tool for osteoporosis or osteopenia.