CDS Symposium

Cartilage Regeneration
The Implications of Microfracture

Date (04/03/2021)

Seeon Chang, Jongpyo Hong, Woogun Noe, Ji Won

Min

1
 CDS Symposium

Abstract

- Short description of the paper

Cartilage, being the connective tissue that cushions the joints, degrades with overuse and

age. With its lack of nerves, the impacted damage to the cartilage cannot be detected with ease.

Such effects have led to high exposure to arthritis and other injuries. Not only that arthritis is

dangerous but also that it is prevalent among adults. Over 23% of adults within the United States

are affected by arthritis. Such a large number proves cartilage’s gravity to the general public.

Currently, the treatment for such damage is not fulfilling its potential and does not have the

ability to heal completely. With the aim of ameliorating cartilage’s function and its implications

to our body, this paper emphasizes and drives for further research into the regeneration of hyaline

cartilage. With already proven effective methods at hand in the past, it is safe to proclaim that the

cartilage has the potential to reform its desired functions to the body when repaired with

discussed techniques.

2
 CDS Symposium

Contents

1. Introduction

1 in 4 Americans over the age of 18 suffer from arthritis and far more suffer from

undiagnosed joint issues from long-term overuse and injury to the joint systems. Despite the

mystery surrounding the regenerative abilities of cartilage, its contents can be understood in

terms of what Assistant Professor of Surgery Charles K.F. Chan, Ph.D., Stanford University, calls

a “STEM cell empowered microfracture.” Let’s say one has suffered a joint injury, such as a

ruptured ligament, whatever it may be, this destabilizes the joint which in turn affects the

cartilage in the long term by wear and tear. This process can take a year or even up to a decade

after injury. However, it is not reversible. The early changes in cartilage are difficult to feel since

there is no neural tissue within the cartilage itself, but after losing most of the cartilage, the bones

become exposed and begin to rub directly against each other. This in turn causes noticeable pain.

Awaiting damage and future research will not be sufficient for a new technique to be possible for

individuals to periodically boost their articular cartilage in advance. Increased medical funding

and attention to the certain limitations of our cartilage must be in order for any approval of an

experimental technique in the future.

3
 CDS Symposium

2. Body 1

There are four types of tissue: neural, epithelial, connective, and muscular. Cartilage is

this connective tissue that constitutes the flexible part of the ear, the end of the nose, but most

importantly, the cushioning of the joints. It is also what the skeleton of animals such as

cartilaginous fish as sharks or rays are formed of. Whether one is an athlete or an avid exerciser,

understanding the basics of cartilage tissue will assist in prolonging one’s career in athletics or

possibly even one’s own lifespan. Cartilage is a connective tissue like bones, blood, fat, and even

tendons and ligaments. But they are easily distinguishable from these as well. If one was to break

a bone or tear a muscle, they would realize as soon as they were to break it as it is full of neural

tissue. However, cartilage does not contain any neural tissue. Thus, one will not be able to notice

if their cartilage has been damaged unless it were to affect the underlying bone. Moreover, those

bones and muscles will heal over time. Cartilage does not, and “needs more robust exogenous

approaches to achieve satisfactory regeneration” (Unlike Bone, Cartilage Regeneration Remains

Elusive, 2012). To further understand how cartilage is crucial in our daily livelihoods as humans,

one must know the three different types of cartilage: hyaline cartilage, which is smooth and rigid,

has minimal flexibility, and a smooth glassy surface which allows the bone to move effectively

within our joints; fibrocartilage which is rough but flexible, located in the intervertebral disc;

elastic cartilage, the “most springy, supple type of cartilage” which adds categorical

differentiation of cartilage which provides support. These are surrounded by cells known as

Chondrocytes. These cells contain a network of threadlike elastic fibers, which allows them to be

flexible but also resilient at the same time. Many individuals who have the basics in

understanding how cartilage functions may believe that cartilage does not heal nor does it grow

back. In truth, it can, but at an extremely slow rate of regeneration. Like other tissues, cartilage is

4
 CDS Symposium

made up of cells. These cells as mentioned previously are known as chondrocytes. When other

tissues get damaged, they are able to migrate to the wound and undergo mitosis rapidly in order

to repair the hole that had been made. However, chondrocytes cannot do this for two reasons.

Firstly, they are found in a thick matrix, in which “the molecular constituents of the cartilage

matrix are laid down into large multi-molecular assemblies” (From Gristle to Chondrocyte

Transplantation: Treatment of Cartilage Injuries, 2015). Thus, they are unable to easily relocate

their positions with their

microtubules. Secondly, there are

no arteries or veins in the

cartilage, meaning that their blood

supply is limited. Consequently,

sufficient nourishment of these

cells with the necessary nutrients

is not available, as cartilage does

not have sufficient access to

“abundant nutrients,” and therefore “lacks innate abilities to mount a sufficient healing response”

(Huey, 2012). The limited supply that they receive is through diffusion. Yet, this is simply not

enough for the rapid onset of mitosis that is required for regeneration to occur. As mentioned

previously, if one has suffered an injury such as a ruptured ligament that destabilizes the joint

which in turn damages the underlying cartilage in the long term by overuse, the bones become

exposed over time. This produces frictional forces between the edges of the bones themselves,

and “can damage newly developed tissues that do not possess required lubrication, compressive,

or tensile properties” (Hu, 2012).” This causes noticeable pain. Initially, this degradation is not

5
 CDS Symposium

usually noticed by the individual; nevertheless, it becomes evident only after years of untreated

orthopedic issues regarding the damaged joints.

6
 CDS Symposium

3. Body 2

Articular cartilage supports the elasticity and the resistance to compressive forces done so

by injuries or traumatic experiences, thereby protecting the more rigid underlying bone and

ensuring smooth articulation at the surfaces. Cartilage has a prominent extracellular matrix due

to the microtubules that support the movement of the chondrocyte cells. This matrix is

categorized as two major constituents: the collagen matrix and proteoglycans. The remainder of

the mass includes other proteins, the functions of which are currently poorly understood in

science, as well as, lipids, nutrients and inorganic substances. The chondrocyte cells are

presumably capable of synthesizing all the constituents required for the formation and

maintenance of the cartilaginous extracellular matrix, and is the “producer of the matrix

components of cartilage” (Lindahl 2015.) It is not known if the cells synthesize all the products

simultaneously or in regulated phases depending upon the requirements of the tissue. Moreover,

the chondrocytes, although sparsely distributed, play a crucial role not only in structuring the

tissue but more essentially in regulating synthetic and anabolic processes which are of

significance in both health and disease propagation. An understanding of the chemical properties

of cartilage requires an in -depth understanding of the nature and structuralization of its

constituent bio-macromolecules.

These molecules change during life, possibly to meet altered functional requirements.

The predominant fibrous component, type II collagen, makes up 40-70% of the dry weight, with

“elastin abundant in elastic cartilage” (Lindahl 2015). It is unique to cartilage (but also present in

7
 CDS Symposium

a few other tissues, e.g. the vitreous body of the eye). It forms a network of thin fibers that

structures the correct shape and determines the maximum volume of the tissue by rendering

useless the vast expansion of the

hydrophilic proteoglycans. Until

recently, type II collagen was

thought to be the only existing

species of collagen in hyaline

cartilage, but additional minor

Fig 1.2 (Histological appearance of articular cartilage and chondrocyte) collagen groups have been reported

to also be present in vitro. Most of these collagens are structurally different and only type IX

collagen is believed to also be a part of the formation of the collagenous network of the cartilage.

The IX is centralized at the intersection of collagen inter-fibrils and may function as a connector

between fibrils of type II, limiting their extensive purposes. The relevance of type IX in the

continued use of a functional collagenous inter-fiber network has been incorporated into a

morphological model portraying the properties of the extracellular matrix. The findings indicate

that the functional structural framework in which the articular cartilage functions may be

significantly jeopardized when the inter-fibril cross-links are degraded. Type XI collagen on the

other hand is located in the interior of type II cartilage collagen fibrils and may be controlling the

lateral growth of the inter-fibrils during fibrillogenesis (synthesis of inter-fibrils within the

collagen matrix) in vivo (within a live organism). Type X collagen is selectively synthesized by

proliferating chondrocytes of the hypertrophic zone in which the matrix is destined to become

calcified and to be replaced by pre-existing bone structures. The proteoglycan groups, entrapped

in the collagen network and making up 15-40% of the dry weight of the cartilage, are molecules

8
 CDS Symposium

with high concentrations of negatively charged sugar groups attached onto transmembrane

proteins that allow for intercellular biochemical signalling between groups of limited red

blood cells and the chondrocyte cells. All proteoglycans consist of a central polypeptide to

which one or more glycosaminoglycans are attached covalently to the long side of the chain.

Glycosaminoglycans are linear anionic

polysaccharides with repeating

disaccharide units containing a

hexosamine residue and usually, but not

always, a hexuronic acid residue. This

allows for the PGs to effectively

communicate through local signalling

between the chondrocyte cells. In the

current model for aggregating PG of Fig 1.3 (Extracellular collagen matrix within the chondrocyte domain)

cartilage, the core protein has at least two, and in some cases, three globular domains. The

primary globular domain, the HA-binding region, is located at the N-terminal end. It was found

by electron microscopy as separated by roughly 21 nm the second globular domain. A third

globular domain is present at the C-terminus. The CS-rich region carries most of the

approximately 100 CS (chondroitin sulfate) chains, which play an essential role of cartilage by

adding to elasticity and function of articular cartilage, and 20-50% of the 20-80 KS (keratan

sulfate) chains, which play a role in glial scar formation following an injury, extend over

approximately half of the core protein. Many non sulfated oligosaccharides are attached to the

core protein of the PG (proteoglycans). These include O-glycosidically linked oligosaccharides

located predominantly in the CS-rich region of the molecule, and N-glyco saccharides located

9
 CDS Symposium

predominantly in the HA-binding domain. It has been researched that proteoglycans contain

phosphate ester groups functionally located on xylose chains within the central amino acid chain

that have an unknown role within the collagen matrix to be researched in the future. Most of the

PGs (proteoglycans) within the cartilage matrix seem to be present as aggregates. PG

(proteoglycan) monomers can interact specifically and non-covalently via their HA-binding

region with a small segment of a single molecule of HA. As many as 200 PG (proteoglycan)

monomers can bind to one single HA molecule 10 6 daltons to form an aggregate of 5(107) to

5(108) daltons that, when examined by electron microscopy, is more than 2μm long. A third

component, the linked protein, stabilizes the attachment of each monomer to HA by interacting

both the HA and the HA-binding region of the core protein. Linked-stabilized aggregates do not

readily dissociate at physiological pH in non-denaturing conditions. The mechanisms involved in

this aggregation process, which has been shown to take place extracellularly, and the assembly of

aggregates within the collagenous network surrounding the chondrocytes are poorly understood.

10
 CDS Symposium

4. Body 3

Currently, further research has exacerbated the replenishment of hyaline cartilage by

inputting small amounts of damage to the joint tissue and the underlying bone small drill holes,

utilizing local signalling to redirect the regeneration of the cartilage stem cells as the damage

heals. At a point in which cartilage is mostly gone from the injured area, trained orthopedics can

only medicate the pain with painkillers. However, if the injury is diagnosed early, encouragement

of cartilage regeneration is possible. The most concurrent technique in which this is done is

through a procedure known as microfracture. This procedure cleaves small crevices within the

underlying bone that penetrates into the bone marrow. However, the new tissue is different from

the cartilage. Microfracture ensues into fibrocartilage, acting as scar tissue and protecting the

bone. Yet, the issue still stands: the required smooth hyaline cartilage that allows effective use of

the joints is not what is produced. Rough fibrocartilage as mentioned before is essentially used as

a scar tissue that assists in the protection of the two underlying bones. However, it is not as

smooth enough as hyaline cartilage in order for rotation to remain relatively with ease, tending to

degrade at a faster rate. Since the goal is the nice smooth cartilage, orthopedics do have one other

advanced procedure in order to attain the hyaline cartilage: they will often attain grafts from

elsewhere on the body or from a cadaver. However, one cannot simply take the cartilage. A small

plug that consists of the bone and the cartilage is taken from the origin. The implanted bone will

heal relatively quickly with the newfound cartilage and hopefully lead to a successful operation.

Yet, the issue with this is that the body may reject the tissue coming from another patient. Even if

this is not the case, the point in which the cartilage was taken from is left with a hole.

The method in which to understand microfracture is through looking at the movement of

the resulting stem cells after the procedure, as the process involves “subchondral bone

11
 CDS Symposium

penetration to release bone marrow that forms a stem cell–rich clot” (Huey 2012). It has been an

underlying assumption since the discovery of cartilage degradation that adult cartilage tended not

to regenerate after trauma due to the lack of stem cells available within the tissue. Despite such a

held belief, it was discovered that this new technique did stimulate skeletal stem cells to

regenerate fibrocartilage in the locations in which the injury had affected the damaged cartilage.

For future repairs, scientists are working on growing the plugs from one’s own cells by taking

adult stem cells from fat cells that could be turned into bone and cartilage, then through various

techniques, signal to those stem cells to differentiate into bone or cartilage. This can be done

through two methods: first with interstitial growth which divides the cells in the collagen matrix

structure and regrows the cartilage from within the bone itself, and second with appositional

growth which divides the cells from the outer surface of the collagen matrix, growing the

cartilage from on top of the underlying bone. Both methods are highly experimental and are far

from being commercially available to consumers. Furthermore, cartilage replenishment in both

cases applies to the mesoderm germ layer through chondrogenesis. It is crucial that this process

occurs within the mesoderm along with red blood cells as sufficient nutrients are required for the

growth. The mesenchyme mesodermal embryonic tissue specializes into newly differentiated

chondroblasts which in turn are the cells that excrete (through the major constituents of the

extracellular matrix’s translocational methods in the form of microtubules. As the initial stages

chondrification occurs, cartilage forms primarily by developing through the G1 and G2 phases of

the cell cycle, as it cannot undergo mitosis. Thus, it is crucial to allow the cells to differentiate

after sufficient time has been passed in the replenishment phase. Additionally, the current state of

methods in which cartilage is regrown is relatively slower than what is planned for the future.

Although currently microfracture is not seen as the optimal technique, it turns out that

12
 CDS Symposium

microfracture can be used towards the development of cartilage instead of fibrocartilage with

further research and funding. The process of proceeding from the cartilage stage is thought to be

able to be hindered by future research with sufficient funding. In order to execute these ideas into

a sound experiment, researchers had used Bone Morphogenetic Protein (BMP2). This initiated

bone formation processes after microfracture. However, the prevention of its continuation with

vascular endothelial growth factor (VEGF) has allowed researchers to get the rudimentary

concepts on how this could be done with more complex mechanisms. This experiment has

resulted in cartilage consisting of cells highly considerable to hyaline cartilage with akin

properties and factors to before the traumatic experience on the injured area. It had also brought

back the mobility to the osteoarthritic mice and decreased the level of indicated pain, thus

“[enhanced] hyaline quality and [increased] fill percentage” (Hu 2012). On the basis that this

may be applicable to human subjects in the future, it is crucial to conduct similar levels of

experiments in animals of larger size prior to human trials. One advantage of the cartilage

regrowth discovery is that it has already been approved by the FDA for effectiveness and is

currently being researched in regards to VEGF inhibitors used as anti-cancer therapies. This new

technique will allow individuals to periodically boost their articular cartilage in advance.

Therefore, it is crucial that future research is conducted in a manner that is proven effective

through an increase in funding.

13
 CDS Symposium

5. Conclusion

Articular cartilage is highly specialized in regards to other types of connective tissue. The

primary role is to provide a surface in which the underlying bones have an efficient method to

rotate about the joints. The properties of this tissue depend on the interaction of its collagen

matrix. However, the structural complexity of hyaline cartilage makes its replenishment a drastic

difficulty. Currently, degraded cartilage can be treated through microfracture in which small

amounts of damage is made onto the underlying tissue of the joint. Microfracture forces the body

to generate new cartilage tissue within the joint; however, this results in the regeneration of the

cartilage in vivo by means of fibrocartilage, which does not have the elasticity of hyaline

cartilage, thus cannot be an effective surface for articulation. Moreover, researchers have

predicted that one day, avoiding arthritis by rejuvenating the cartilage in the joints before it gets

significantly degraded is a possibility in the future. Instead of awaiting the for the damage to take

place in the body, this new technique will allow for a periodic boost for their articular cartilage in

advance. This will possibly even pathe the way for the regeneration of smooth hyaline cartilage

in the future with microfracture techniques.

14
 CDS Symposium

6. Bibliography

"Arthritis." Mayo Clinic. Mayo Foundation for Medical Education and Research, 19 July 2019.

Web. 02 Apr. 2021.

Bentley, George, and Tom Minas. “Science, Medicine, And The Future: Treating Joint Damage

In Young People.” BMJ: British Medical Journal, vol. 320, no. 7249, 2000, pp.

1585–1588. JSTOR, www.jstor.org/stable/25224776. Accessed 2 Apr. 2021.

Carter, Dennis R., and Marcy Wong. “Modelling Cartilage Mechanobiology.” Philosophical

Transactions: Biological Sciences, vol. 358, no. 1437, 2003, pp. 1461–1471.

JSTOR, www.jstor.org/stable/3558281. Accessed 2 Apr. 2021.

Huey, Daniel J., et al. “Unlike Bone, Cartilage Regeneration Remains Elusive.” Science, vol.

338, no. 6109, 2012, pp. 917–921., www.jstor.org/stable/41703940. Accessed 2 Apr.

2021.

Lindahl, Anders. “From Gristle to Chondrocyte Transplantation: Treatment of Cartilage

Injuries.” Philosophical Transactions: Biological Sciences, vol. 370, no. 1680, 2015, pp.

1–7., www.jstor.org/stable/24505277. Accessed 2 Apr. 2021.

Malda, Jos, and C Wayne McIlwraith. "Current Trends in Cartilage Science: An Impression from

the ICRS World Conference 2012." Cartilage. SAGE Publications, Oct. 2013. Web. 02

Apr. 2021.

Morrison, William. "Cartilage Damage: Symptoms, Causes, Diagnosis, and Treatment." Medical

News Today. MediLexicon International, 15 Dec. 2017. Web. 02 Apr. 2021.

15
 CDS Symposium

Pak, Jaewoo, Jung Hun Lee, Wiwi Andralia Kartolo, and Sang Hee Lee. "Cartilage Regeneration

in Human with Adipose Tissue-Derived Stem Cells: Current Status in Clinical

Implications." BioMed Research International. Hindawi, 06 Jan. 2016. Web. 02 Apr.

2021.

Park, Jinhyung, et al. “A Simple in Vitro Culture System for Tracheal Cartilage Development.”

In Vitro Cellular & Developmental Biology. Animal, vol. 46, no. 2, 2010, pp. 92–96.

JSTOR, www.jstor.org/stable/40602787. Accessed 2 Apr. 2021.

Parvizi, Javad. "Cartilage." Cartilage - an Overview | ScienceDirect Topics. ScienceDirect. Web.

02 Apr. 2021.

Saintigny, Yannick, et al. “Impact of Therapeutic Irradiation on Healthy Articular Cartilage.”

Radiation Research, vol. 183, no. 2, 2015, pp. 135–146., www.jstor.org/stable/24545470.

Accessed 2 Apr. 2021.

16