Chondrocytes are cells found in cartilage that secrete collagen and proteoglycans to form the cartilaginous matrix. There are three types of cartilage - elastic, fibrocartilage, and hyaline cartilage - containing different numbers of chondrocytes. Chondrocytes play a key role in endochondral ossification, a process by which bones grow and mature. During this process, chondrocytes proliferate, mature, and eventually die to be replaced by osteoblasts that lay down new bone. The differentiation of mesenchymal stem cells into chondrocytes is regulated by signaling factors such as BMP and Sox9. Chondrocytes have low regener

1. Chondrocyte Cell Profile Report
Introduction
During human fetal and postnatal development, chondrocytes facilitate bone
formation via endochondral ossification, and continue to provide support, structure
and flexibility in the adult1. These cells make up about 10% of cartilage, a loose
connective tissue found in bone joints, bronchial tubes, the ears and nose, and are
responsible for secreting collagen and proteoglycans that make up the cartilaginous
matrix2. The composition of this matrix varies depending on the type of cartilage and
its function3. Of the three varieties, elastic cartilage contains the highest number of
chondrocytes, is the most flexible and makes up the structure of the outer ear. There
are far fewer chondrocytes present in the less flexible fibrocartilage, which provides
support between the vertebrae, at the knee joint and in the pubic symphysis. Hyaline
cartilage is found at the end of long bones and in the nose, containing fewer
chondrocytes than elastic cartilage, but more than fibrocartilage2.
Chondrocytes provide the cartilaginous scaffold for endochondral ossification – a
process fundamental to the longitudinal growth of bone4. Endochondral ossification
occurs in the human embryo, and continues in postnatal development until bones have
fully matured1. During this process, chondrocytes proliferate, mature and die, before
they are replaced by osteoblasts, which build bone2. Cartilage engaging in
endochondral ossification is referred to as growth cartilage, and is found at the

2. primary and secondary centres of ossification: the diaphysis and epiphyseal growth
plates, seen in Figure 1.11. Chondrocytes in growth cartilage can be observed moving
through separate zones (see Figure 1.2), indicating which stage of endochondral
ossification they are in. Resting chondrocytes are round in shape, surrounded by
extracellular matrix (ECM) and are most remote from the ossification centre. Bone
morphogenic protein (BMP) signaling drives proliferation of chondrocytes1, which
become flattened in shape, and can also be characterized by their expression of Sox5,
Sox6 and Sox95. Adjacent to the zone of proliferation are pre-hypertrophic
chondrocytes, which secrete Indian hedgehog (IHH) to aid both proliferation and
hypertrophy1. These chondrocytes then increase about 20 times their original size6,
and are referred to as hypertrophic, and are responsible for mineralizing the ECM
surrounding them. Finally, the chondrocytes die (likely via apoptosis7) and part of the
ECM is removed, allowing the entry of osteoblasts to construct new bone4. Figure 1.1
shows that hyaline articular cartilage remains at the end of long bones, aiding in
lubrication, support and movement of joints.
To initiate human embryonic development, two gametes (one sperm and one oocyte)
must fuse to form a diploid cell – a process known as fertilization8. This single cell
undergoes mitotic division, producing new cells referred to as blastomeres. By the 16-
cell morula stage, there are already transcriptional differences between the cells,
although they are still totipotent. 5 days post-fertilization, a blastocyst has been
formed, showing a distinct structure called the inner cell mass. This delaminates into
dorsal and ventral layers: the epiblast, giving rise to embryonic tissue; and hypoblast,
which becomes extra-embryonic structures. All cells, with the exception of the germ
line, are derived from the germ layers formed during gastrulation2. The formation of
the primitive streak indicates the beginning of this process, and involves the
condensation, proliferation and migration of epiblast cells in a linear fashion8. As
cells move through the elongating primitive streak, they change shape and begin to
form the endoderm, mesoderm and ectoderm germ layers. The paraxial mesoderm is
located in the anterior region of the primitive streak, and gives rise to brick-shaped
bundles of mesenchymal cells, referred to as somites. Within the somites, signaling
molecules control gene expression, leading to the formation of dermatome, myotome
and sclerotome layers. The sclerotome is created as a result of sonic hedgehog and
noggin, released from the notochord. These molecules increase the expression of Pax1
and Pax9 in the somite’s ventral region, leading to cell proliferation, a loss of N-

3. cadherin and the conversion of proximal epithelial cells to a mesenchymal
morphology8. These cells secrete molecules found in cartilaginous matrix, such as
chondroitin sulfate proteoglycans, and aggregate near the notochord. Although a few
key regulatory steps leading to chondrogenesis have been discovered, the cellular
processes that influence the differentiation of mesenchymal cells to chondrocytes are
mostly unknown5. Inhibition of retinoid signaling is required to stimulate Sox9
expression in prechondrogenic mesenchymal cells – a key step in chondroblast
differentiation9. Chondroblasts are more spherical in shape, and begin to secrete the
cartilaginous matrix. They become trapped in spaces filled with extracellular fluid,
called lacunae. Once the chondroblast is surrounded by the lacuna, it is considered a
mature chondrocyte. Depending on its location and the host organism’s stage of
development, the chondrocyte can either become part of the permanent cartilage, or
can undergo endochondral ossification8. The linage of the chondrocyte from
mesenchymal cells, and the signaling factors involved in differentiation, is described
in Figure 1.3.
Chondrocytes have
low reparative and
mitotic capabilities, as
cartilage is avascular3.
The exchange of
nutrients and waste
products is therefore
slow, and occurs by
diffusion from blood
vessels and synovial
fluid10. It is
consequently critical
to understand the
processes involved in
chondrogenesis, so that disease of the cartilage may be prevented or treated
appropriately. Osteoarthritis, for example, is a degenerative disease of the joints,
caused by overuse or injury of the cartilage11. Globally, it is the most widespread
musculoskeletal disease12, extremely prevalent in the elderly and presenting a huge
economic burden3.

4. Results
Figure 2.1 shows the complete section of a mouse embryo, including the vertebral
body and ribs undergoing endochondral ossification. Bone shown in the limb has
already undergone this process. Cartilage is found in different locations in the adult.

5. Figure 2.2 illustrates a closer view of the vertebral body (stained blue) undergoing
endochondral ossification.
At this magnification the different zones of endochondral ossification at the vertebral
body can be observed. From the zone of resting cartilage, chondrocytes move towards
the zone of ossification as they proliferate and hypertrophy.

6. From 20x magnification it’s possible to discern the hypertrophic chondrocytes from
those undergoing apoptosis. The ECM is becoming mineralized to allow osteocytes to
migrate to this region and lay down new bone. A secondary site of ossification can
also be seen, in which chondrocytes are undergoing hypertrophy but have not yet
undertaken cell death.
In figure 2.5 the nuclei (purple) and lacunae (white) of the chondrocytes are visible.
The cells are suspended in the ECM (blue), composed of collagen, proteoglycans,
fibres and water.

7. Discussion
In the macromolecular justice system, the cells are represented and protected by two
separate but equally important groups: the B-cells, who investigate pathogens; and the
T-cells, who prosecute the offenders. This, however, is not their story. (Click here for
rad interactive reader experience).
In the distant district of hyaline cartilage, the leukocyte law and order team rarely
show their faces. In the beginning, fibroblast growth factor 2, vascular endothelial
growth factors and angiopoietins cared not to build roads for chondrocytes. They rely
on diffusion (an ancient and inefficient mode of transport) for the delivery of nutrients
and waste disposal10. In this low oxygen environment, lived one peculiar chondrocyte,
known as Chonelius. As a young mesenchymal cell, Chonelius grew up listening to
stories conserved from ages past, remembered by the those who had not gone on to
differentiate. They told the bleak history of the cartilaginous scaffold gang war, which
sadly ended in endochondral ossification4. Countless chondrocytes fearlessly
hypertrophied, but their bodies were left scattered, and the osteoblasts were
victorious. Some chondrocytes were in the right place at the right time, and managed
to survive, but those who were left carried a larger burden. The bones have been in
power ever since, their density supported by the tireless work of chondrocytes. They
didn’t seem to care that bone and cartilage were distant brothers8. Sox9 eventually
contacted Chonelius. Accepting his fate, he underwent a morphological change and
became a chondroblast13. He knuckled down and began to secrete fibres, collagen and
proteoglycans, which were continually compressed under the weight of bone. Soon he
had surrounded himself in his very own lacuna8, and all who knew him celebrated his
coming of age. He was named a chondrocyte; a tough cog in the machine, built for
survival.
Chonelius, however, was not an ordinary chondrocyte. He noticed a shift in the
matrix, feeling there were not quite as many of his kind as there once was14. It wasn’t
always easy to talk to his friends about it, as so much cartilage came between them,
but when he managed to mention it, they were skeptical. “Lacuna matata”, they told
him, “times have been worse”. Chonelius was not reassured. He was sure
chondrocytes were disappearing, and suspected the bones had a hand in it, but it was
near impossible to communicate with the feds3. It was up to him to get to the bottom
of this.

8. Word on the street had it that Chonelius’ suspicions were sensed in other cartilaginous
hoods. He smelt death in the air. Over the months, some of Chonelius’ friends became
distant, while others disappeared. The matrix was wearing thinner, slowly losing the
capacity to support the ominous femur. The world began to creak and groan in
despair. Rogue cytokines were becoming more common in these parts, inflaming the
already dire situation15. This was more than a simple murder mystery case. This was
genocide, and the bones were getting away with it. After a few days dealing with
cytokines, Chonelius noticed some strange molecules he’d never seen before. They
seemed to know the pro-inflammatory cytokines well. Chonelius recognized his
chance to solve the mystery. Slowly, he crept behind one of the fresh faces, cuffed his
hands and took him in for questioning. Chonelius was no bad cop. Thankfully he
didn’t have much trouble getting the molecule to talk. His name was Chondroitin
Sulfate, a drug lord from another universe, who was apparently trying to help. He’d
had quite a journey from “the lab”, to packaging and through the gastrointestinal
district to get here. Chondroitin Sulfate didn’t seem to have much more information,
so Chonelius let him go. Chondroitin Sulfate and his friends seemed to help for a
couple of weeks, but Chonelius heard the gut was nauseous and the brain was
experiencing vertigo16, so they, too, dissipated.
Chonelius ramped up his global networking. He heard skeletal muscle was
atrophying, while adipocytes proliferated and sat greedily idle, exacerbating the
inflammation they all endured. Astonishingly, there was a rumour that even the bones
had begun to lose their density. Chonelius was in shock. How could his prime suspect
be suffering? If this were true, the case was much larger than Chonelius could handle
alone. He pushed aside his skepticism, and turned to the powers that be to put an end
to this epidemic. His eyes
closed, and his prayers went
unanswered.
The slow rumble of the bones
roused Chonelius the next
morning. He had barely opened
his eyes, when there erupted an
earsplitting sound he had never
heard before; osteoblasts and
osteoclasts screaming in their thousands, grinding against each other17 as the matrix

9. was pushed to the side. Then, from the heavens above, all the cells heard a majestic
voice … “Ouch!” it said. Chonelius furrowed his brow. He had hoped for a little more
clarity.
The screaming endured for months. Chonelius and the other survivors were working
overtime, but were unable to produce enough cartilage. The mesenchymal cells were
doing their best to migrate12, but could not proliferate and differentiate at the rate they
once did18. Chonelius began to brainstorm. Perhaps there was a way to develop more
chondrocytes in that “lab” Chondroitin Sulfate spoke of, and transplant them here19?
What if he could get a message to the powers that be to send more mesenchymal stem
cells20? If the cartilage cannot be repaired or replaced, was there are least a way to
calm the nociceptors down?
Just as Chonelius was pondering about the nervous system, there were sounds of
distant confusion, then silence. A chemical had entered the system, and caused the
neurons to decrease their activity. The skeletal muscle stopped contracting, easing the
pressure on the cartilage, but a steady thudding carrying from the cardiomyocytes to
the smooth arteriolar muscle could still be heard. It was like a strange, unexpected
sleep; the world was eerily still. The cells were uneasy, and Chonelius couldn’t help
but feel something big was on the way. He nestled in his lacuna, feeling more alone
than ever. The screams from the epithelial cells started first. Suddenly, a wave of
erythrocytes, leukocytes and plasma came flooding into the cartilage, and for the first
time since becoming a chondrocyte, Chonelius was mobile. A chaotic crowd of
panicking cells surrounded him, many of them injured or dead from the fallout. A
whirring sound began overhead, terrifying the cells into silence. A giant piece of
spinning metal appeared, grinding through the top of the almighty tibia beneath them.
The metal made its way from one side, all the way to the other, until the top of the
bone was completely removed from the rest. Chonelius could barely watch the bloody
massacre. He prayed for it to be over, for it to be a dream. But instead, gravity shifted,
as rubber grasped the butchered bone and pulled upward and outward. Photons
whizzed past, bouncing off Chonelius and back to the iris of a giant. Masked,
goggled, her rubber hands moved Chonelius’ home to a table full of instruments.
Chonelius watched her push the metal through the tip of the femur, remove it, and
measure up her loot17. On the table next to him were two more pieces of metal, one
similar in shape to the piece of femur. The giant took the metal, turned her back and
inserted it where the bone once was. Chonelius couldn’t believe what he was seeing.

10. His gaze adjusted, affording him more perspective. There, on the table, lay another
giant. The femur, the tibia, belonged to this giant! God almighty! All wrinkly3!
Chonelius realized he had met his maker, and he was at war with a masked giant. The
end was nigh, but the case was out of little Chonelius’ hands. He closed his tired eyes,
hoping at long last justice would be served.
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