Bone tissue formation

Hey everyone! Nicole from Kenhub here, and today we're going to be talking about bones!

No doubt you'd be aware that your bones are pretty unique body tissue. They allow you to move and stay upright as well as produce vital blood cells in their marrow. But have you ever wondered how your bones grow and form in the first place? In this tutorial, we'll be diving right into the bones at their cellular level and looking at the histology of bone tissue formation.

Bone formation, also known as osteogenesis, refers to a complex series of interconnected processes like cell migration, multiplication, differentiation as well as synthesis and excretion of extracellular matter and cell degeneration. We can differentiate between two types of bone formation based on the initial histological environment in which a bone begins to form, and these are intramembranous ossification and endochondral ossification.

So, we're going to be looking at each of these processes in turn starting with intramembranous ossification. Intramembranous ossification is the formation of bone through the clustering and differentiation of mesenchymal stem cells around highly vascularized connective tissue. And these cells gradually differentiate into more mature and specialized cells which we'll talk about in more detail in just a little bit.

So if we look closely at our image, we can identify several distinguishing histological features unique to this type of ossification but first, let's orientate ourselves. Our histology slide here is taken from a section of fetal skullcap and first of all, we can clearly see several blood vessels which indicate the highly vascularized nature of the connective tissue just here, and this border of blue stain is known as osteoid which is the unmineralized portion of the bone matrix and which we'll talk a little bit more about later. And these areas of deep red are a final product of intramembranous ossification which is mineralized bone. And that the distinguishing feature which is very prominent in our histological slide is tightly packed collagen, and this can be expected, of course, in connective tissue.

This collagen forms structures known as trabeculae which we'll also discuss a little bit later but for now, let's focus on the specific cells involved in the intramembranous ossification.

So remember that we talked about mesenchymal cells differentiating into other cells. We're now seeing the product of this differentiation. These cells are known as osteoblasts and they originate from mesenchymal stem cells. You can see that they cover these collagenous structures we previously presented but their role in bone formation is much more complex than that. These cells basically synthesize and secrete different substances, each with their own importance in the process of bone formation.

First of all, they produce all the collagen which organizes into the collagenous structures we presented, specifically type one collagen. And an easy way to remember this is that type one collagen is contained within bone. Another substance produced by osteoblasts is proteoglycan. Type one collagen together with the proteoglycan constitute a matrix for the newly formed bone which is known as osteoid. And finally, osteoblasts produce and secrete alkaline phosphatase. This is an enzyme which promotes the calcification of the osteoid matrix.

Let's now look a little bit more closely at the osteoid matrix which, if you remember, is composed of type one collagen and proteoglycan and is calcified by alkaline phosphatase. New osteoid forms on preexisting mineralized osteoid so osteoid is laid down layer by layer, and this process results in the formation of a matrix which is initially soft and pliable and hardens once it calcifies. This matrix initially calcifies into small bony islets which later grow to form the bony trabeculae and, eventually, the bony trabeculae thicken and connect to each other to form what is later known as trabecular bone.

The space between the trabeculae accommodates the highly vascularized hematopoietic tissue which is known as the bone marrow, and as you can see, many osteoblasts become trapped in small cavities within the calcifying osteoid and these cavities are known as lacunae, which is the Latin name for pit.

The osteoblasts differentiate one step further to become osteocytes and these cells sense mechanical stress on bone and can secrete growth factors that stimulate osteoblasts in response to that stress. They're also believed to transfer minerals from the interior of bone to the growth surfaces. Our next cell type is formed by the fusion of macrophages making it multinucleated. These are known as osteoclasts, and we can see some on our histology slide just here. Do take a note their foamy appearance as this a good way to identify them.

The cytoplasm of osteoclasts contains a high concentration of vesicles and vacuoles and these vacuoles include lysosomes filled with acid phosphatase. Osteoclasts move along the surface of the bone and seal themselves to the bone through integrins binding to fibronectins on the bone surface and by emptying their lysosomal contents of acid phosphatase on the bare mineralized osteoid permitting bone resorption.

Osteoclasts are not located just anywhere on the osteoid matrix of bone. They can be found specifically in pits in the bone surface which are called resorption base or Howship's lacunae. And bear in mind that not every osteoclast in these lacunae participates in active bone resorption. If we look a little bit more closely at our image, we'll see a large orange cell and this is the osteoclast near the surface of the bone. We can see that the connective tissue near the bone has been thinned out as is the osteoid underlying it.

At another site of active bone resorption, we can see the osteoclasts forming what is known as the ruffled border which is a specialized cell membrane that opposes the surface of the bone tissue. This extensively folded or ruffled border facilitates bone removal by dramatically increasing the cell surface for secretion and uptake of the resorption compartment contents.

So that's it for intramembranous ossification. Now let's take some time to have a look at endochondral ossification.

So endochondral ossification begins with the formation of a mold made of hyaline cartilage along which ossification occurs and bone forms. These cartilaginous molds take the shape that the bone will have once the process of ossification is complete. In this histology slide of a fetal elbow joint just here, we can see endochondral ossification in action. So, first of all, you can see a large portion of hyaline cartilage occupying much of the image and during intrauterine life, the first part of such bones form is the bone diaphysis which forms as a cartilaginous mold or model. The cartilaginous mold undergoes hypertrophy and mineralization and once this is done, osteoblasts migrate into this area and lay down osteoid around the calcifying cartilage.

Meanwhile, the cartilaginous mold grows longitudinally so that in longitudinal bones, we get to see cartilage cells being organized in columns on either end of each longitudinal bone. These regions on either end of the longitudinal bones are known as epiphysis and another ossification center forms in these regions until puberty. Between the epiphyseal ossification center and the diaphysis is a cartilaginous region which remains until late puberty and this ensures the growth of the bone, and this region is known as the epiphyseal plate.

Let's take a look at another histology slide where we can get a really good look at the epiphyseal plate. So just to reorientate ourselves, this side is the diaphysis and the other side over here is the epiphyseal ossification center.

So the epiphyseal plate is where endochondral ossification takes place in postnatal life. Cells in this region are organized in columns and in four distinct zones which differ in their proliferative activity and we'll look a little bit more closely at these zones in just a moment.

So over on the outer edge of our slide, we can see a structure known as the bone collar. And this ensures that bone will only grow longitudinally and not expand outward and create a misshapen bone. The bony collar is initially formed as cartilage around the bone and then it ossifies and then its perichondrium turns into periosteum.

Alright, let's keep looking at the process of endochondral bone formation and as we do, the first zone we can see and we'll discuss will be seen just here, and this is known as the zone of reserve cartilage. So this region is very near to the epiphysis of longitudinal bones and constitutes of small groups of cartilage cells arranged mostly at random. And as you can see, it also closely resembles hyaline cartilage.

Just below the zone of reserve cartilage, we'll find the zone of proliferation and this region consists of chondrocytes that undergo mitosis regularly under the effect of growth hormone. Therefore, we generally expect this zone to be most mitotically active in childhood and in early puberty. And as we can see in our slide, daughter cells in this zone are arranged in a column-like fashion like a column of coins and arranged in parallel to the longitudinal axis of the developing bone.

Our next zone here is known as the zone of maturation and hypertrophy, and this is where chondrocytes stop dividing and begin to grow in size by collecting lipids, glycogen and alkaline phosphatase intracellularly.

Right under the zone of maturation and hypertrophy lies the zone of calcified cartilage, and this is the zone where the overgrown chondrocytes begin to die over the tragedy leaving behind cavities filled with their waste products which then calcify. Mesenchymal cells from the adjacent bone marrow also invade this area and start differentiating into osteoblasts. The osteoblasts turn the zone of calcified cartilage into a primary ossification center. As we can see, this zone is rich in blood vessels. This is again where the osteoblasts lay down osteoid which in this case builds around the preformed structures of calcified cartilage.

And now last but not least, let's take a look at the bone marrow highlighted here in green. As you can see, there are a lot of blood vessels here as well as collagen trabeculae. We also have some voided areas which we can presume to have been occupied by fat and also cell-rich niches. And all these signs are what you need to be looking at for when you're identifying bone marrow.

So that's it for endochondral ossification. Let's now take a look at some important clinical notes relating to both of the types of bone formation we've looked at.

So to highlight the importance of osteoclasts in their formation of bone, we would only need to look at a condition known as rickets and we can see an example of this case of rickets in this x-ray image. So you may have heard of rickets before. Rickets is a pediatric disease caused by a deficiency of vitamin D in which the bone contains abnormal amounts of unmineralized osteoid and affected individuals are therefore more prone to bone factures.

So we're currently looking at a lower limbs' x-ray of a two-year-old rickets sufferer and if we take a look at the femoral bone, we can see that it has a bended shape towards its lower end and this sign is known as genu varum or the bowing of the femurs. A person with experience in x-ray interpretation would also notice decreased bone opacity which suggests poor bone mineralization. And at this point, you might be wondering how vitamin D, osteoclasts, unmineralized osteoid, and susceptibility to bone fractures are all connected. So, let's find out.

So osteoclasts contain a vitamin D sensitive receptor on their cell membrane which when activated by vitamin D promotes bone resorption. Bone resorption causes calcium that was trapped into mineralized bone to become bioavailable and so becoming available to use in the mineralization of newly formed osteoid.

So remember that osteoblasts lay down osteoid based on osteocyte response to mechanical stress. So we can assume that new osteoid is formed in areas that require mechanical enhancement. But if that osteoid is not mineralized then it can't provide maximal support. So when vitamin D is deficient, that osteoid which was formed in order to better support the bone cannot become mineralized and cannot obtain the same mechanical qualities as the adjacent bone. The result is a bone more susceptible to fracture or attaining unnatural shapes like the bowing of femurs we see in our image.

Our next clinical note is all about achondroplasia, which is an autosomal dominant disorder caused by the mutation in the fibroblast growth factor receptor three gene also known as the FGFR-three gene. When this receptor normally binds fibroblasts growth factor, it makes chondrocytes of the growth plate proliferate at a slow rate and disrupts their columnar organization. Overactivity of this process will therefore affect mostly endochondral bone formation which is exactly what happens in achondroplasia.

The mutated FGFR three gene results in a receptor that is constantly active so it behaves like when it binds in FGF even when it doesn't. So you can imagine that a mutation in the FGFR three gene would result in slower bone growth due to the disruption of endochondral bone formation and you can probably also imagine that since endochondral bone formation is much more prominent in long bones, it would be bones like the femur, the humerus and the phalanges and others that would be affected.

So flat bones like the skull and the ribs depend much less on endochondral bone formation because as they are formed and maintained by intramembranous bone formation which we talk about throughout our tutorial earlier. So a person with FGFR three mutation might have trouble in the growth of his longitudinal bones while his flat bones like the skull and the ribs would grow normally. Achondroplasia results in the commonly perceived phenotype of dwarfism where the person affected grows to adulthood with adult sized flat bones but limited longitudinal growth of long bones.

So, that's everything covered for this tutorial. Let's quickly recap.

In this tutorial, we looked at the histology of bone formation of which there are two types and these are, of course, intramembranous ossification and endochondral ossification. In intramembranous ossification, early bone forms in the form of trabeculae around the blood vessels and this process is the result of osteoblasts secreting and calcifying osteoid in conjunction with osteoclasts contained within lacunae breaking bone down.

In endochondral ossification, cartilaginous molds are formed during embryonic life and turn into bone. We focused on what goes on in epiphyseal plates of longitudinal bones and identified four zones with four different roles in ossification. We specifically presented the zone of reserve cartilage which mostly resembles hyaline cartilage, the zone of proliferation where chondrocytes divide and multiply, the zone of maturation and hypertrophy where chondrocytes grow while being organized in a column-like fashion and, finally, the zone of calcified cartilage where overgrown chondrocytes calcify and sadly die.

Finally, we covered two conditions related to these two types of bone formation which were, of course, rickets and achondroplasia.

So now we finished our summary and our tutorial. Thanks for watching and happy studying!