Maarten Janssen

Life expectancy has increased worldwide and as a way to improve quality of life,
physical exercise is recommended, even at an older age. The positive effects of exercise
have been extensively described in the literature. Ironically, mechanical trauma due to
exercise is described as the biggest cause of focal articular cartilage defects. When
damaged, articular cartilage possesses little regenerative capacity and is prone to
continued degeneration. In the process of articular cartilage damage and joint
degeneration, an important component is the altered expression of biomolecular
factors affecting the homeostasis of articular cartilage and increasing unfavourable
endochondral ossification (EO) of the cartilage tissue. The work presented in this thesis
focusses on understanding alterations in the role of EO in focal and diffuse articular
cartilage damage and possibilities to use the biomolecular mechanisms that are active
in EO for the treatment of articular cartilage damage.

In the first part of this thesis, the influence of patient characteristics on the outcome of
cartilage repair surgery and the role of EO in this process was investigated. In Chapter
2, we analysed patients 25 years after their perichondrium transplantation (PT) and
found that the risk of failure (receiving major revision surgery) was lower and knee
function (measured by International Knee Documentation Committee (IKDC)-score) was
better when adequate patient selection was applied. Factors that significantly changed
the risk of failure in our patient study population were: prior surgery to the index knee
and a longer time of symptoms prior to cartilage repair surgery. A younger age at the
time of surgery was associated with a better IKDC-score at 25 years follow-up. A
morphologic and biochemical evaluation of the cartilage repair by 7T MRI was
performed in patients from the same PT-cohort and compared to a cohort of
autologous chondrocyte transplantation (ACT) patients with a similar follow-up time in
Chapter 3. We found no correlation between clinical questionnaires and the MOCART
(Magnetic Resonance Observation of Cartilage Repair Tissue) score or biochemical
impairment of the grafts. Intralesional osteophytes were common in both the PT and
ACT patients. These osteophytes can result in biochemical damage to the opposing
tibial cartilage. This was more dependent on osteophyte morphology (i.e., an
osteophyte extending into the surface of the graft) than the amount of calcification of
the graft.

In the second part of this thesis, it was investigated how the process of EO can be
influenced. In Chapter 4, we showed that Cyclo-oxygenase-2 (COX-2) inhibition not only A
impaired endochondral ossification in the growth plate and fracture callus, but also in
periosteal cartilage formation in an in vivo rabbit model. The inhibition of COX-2 was
achieved by the use of celecoxib, a selective COX-2 inhibiting drug that belongs to the
class of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). Supported by previous work
from our group that showed a bi-phasic pattern of COX-2 expression in EO, these
results indicate that celecoxib impairs not only hypertrophic differentiation, but also

Addendum

the chondrogenic phase of EO in specific circumstances. Another way to influence the
process of EO was investigated in Chapter 5. The engineering of cartilage tissue from
progenitor cells is often hampered by unwanted EO. To overcome the problem of
adverse chondrocyte hypertrophic differentiation, a previously used biogel to induce
ectopic cartilage formation was supplemented with the major cartilage proteins
aggrecan and cartilage oligomeric matrix protein (COMP). We found that these proteins
were able to improve the process of ectopic cartilage formation in an in vivo rabbit
model by suppressing hypertrophic differentiation of the engineered cartilaginous
tissue.

In the final part of this thesis, possible treatment options were explored to improve the
intra-articular environment in order to reduce joint pain and to postpone or prevent
progression of cartilage damage into osteoarthritis (OA). Currently available oral drug
therapy has many disadvantages that can be overcome by intra-articular administration
of appropriate drugs. However, drug residence time in the joint is generally short and a
drug delivery system (DDS) is needed to improve the efficacy of intra-articular therapy
for cartilage damage. Different types of DDSs are: prolonged release systems,
controlled release systems, and autoregulatory systems. The literature review
performed in Chapter 6 showed that DDSs can be made from different materials. Since
the use of polymers for the development of DDSs, much progress has been made. The
use of DDSs for the treatment of OA is promising, but a disease-modifying combination
of a DDS and drug has not yet been demonstrated in clinical trials. Chapter 7 of this
thesis describes the development and testing of the polyester amide-celecoxib PEA-
CXB-microsphere as a DDS. Pharmacokinetic properties and response to an
inflammatory (OA) environment was tested in vitro in cell lysates obtained from a
neutrophil-like Hl-60 cell line. Subsequently, biocompatibility and degradation of the
PEA-CXB microspheres were tested in an in vivo rat model where the anterior cruciate
ligament was transected and a partial medial meniscectomy was performed to induce
post-traumatic OA. The PEA-microsphere caused no adverse reactions and was found
suitable as a DDS. The PEA-CXB-microsphere did not show an OA disease-modifying
effect. However, increased degradation of the microspheres was present in OA knees,
whereas celecoxib loading of the microspheres reduced microsphere degradation,
suggesting a DDS with an auto-regulatory behaviour. In Chapter 8, key findings of this
thesis were put in perspective.

The worldwide burden of OA is rising rapidly, especially in relatively young patients.
When total knee arthroplasty is performed in young patients, they are at increased risk
for revision arthroplasty later in life. Therefore, more attention for joint preserving
strategies is needed. For successful joint preservation, a timely recognition, patient
selection, and adequate treatment of cartilage damage and OA are needed. Adequate
treatment can be achieved by lifestyle, pharmacological, and surgical interventions and
combinations thereof. The work presented in this thesis demonstrates that the
improvement of joint preservation can be addressed at these different levels and from

different viewpoints/insights (e.g., cell and patient). However, most important for
successful joint-preserving treatment of patients with joint damage is a change in the
approach to the clinical problem by orthopaedic surgeons, other healthcare providers
and scientists. An approach in which the ‘patient journey’ of a person with a healthy
joint can be followed and guided by the right interdisciplinary collaboration up to joint
replacement surgery. This provides the right care at the right time and place to keep
people and patients moving and thus prevent other chronic diseases.

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Addendum

De wereldwijde levensverwachting neemt toe en ter verbetering van de kwaliteit van
leven wordt lichaamsbeweging geadviseerd, zelfs op gevorderde leeftijd. De positieve
effecten van lichaamsbeweging zijn uitgebreid beschreven in wetenschappelijke
literatuur. Ironisch genoeg is een mechanisch trauma bij sporten beschreven als de
grootste oorzaak van lokale kraakbeendefecten. Kraakbeen heeft weinig regeneratieve
capaciteit en een beschadiging van kraakbeen leidt daardoor vaak tot verdere
degeneratie. Een belangrijke component in het proces van articulaire kraakbeenschade
en degeneratie van het gewricht is de veranderde expressie van biomoleculaire
factoren. Deze biomoleculaire factoren beïnvloeden de homeostase van het gewricht
en zorgen voor een toename van ongewenste endochondrale ossificatie (EO) van het
articulaire kraakbeen en osteofyt vorming.
Het werk in dit proefschrift focust op het begrijpen van veranderingen in de rol van EO
in focale en diffuse schade aan articulair kraakbeen en op de mogelijkheden om gebruik
te maken van de biomoleculaire mechanismen die actief zijn in EO voor de behandeling
van beschadigd articulair kraakbeen.

In het eerste deel van dit proefschrift werd de invloed van patiëntkarakteristieken op
het resultaat van kraakbeenchirurgie onderzocht, met ook de rol van EO in dit proces.
In Hoofdstuk 2 werd een groep patiënten 25 jaar na perichondrium transplantatie (PT)
geanalyseerd, waarbij perichondrium van de rib getransplanteerd werd naar de knie.
We ontdekten dat het risico op falen van deze ingreep (gedefinieerd als het ondergaan
van een grote hersteloperatie) lager was en dat de functie van de knie (gemeten door
middel van de International Knee Documentation Committee (IKDC) score) beter was
wanneer adequate patiënt selectie werd toegepast. Factoren die het risico op falen
significant verhoogden, waren een eerdere operatie aan dezelfde knie en een langere
duur van de symptomen voorafgaand aan de kraakbeenoperatie. Een jongere leeftijd
ten tijde van de operatie werd geassocieerd met een betere IKDC-score na 25 jaar
follow-up. Een morfologische en biochemische evaluatie van het herstelde kraakbeen
werd door middel van een 7 tesla (7T) MRI verricht in patiënten van hetzelfde PT cohort
en de resultaten werden vergeleken met een cohort patiënten die in dezelfde periode
een autologe chondrocyten transplantatie (ACT) hadden ondergaan (Hoofdstuk 3). Er
werd geen correlatie gevonden tussen klinische vragenlijsten en de MOCART (Magnetic
Resonance Observation of Cartilage Repair Tissue) score of biochemische verslechtering
van de kraakbeentransplantaten. In zowel PT als ACT patiënten kwamen intralesionale
osteofyten frequent voor. Deze osteofyten kunnen leiden tot biochemisch aantoonbare A
schade aan het tegenoverliggende tibiale kraakbeen. Deze schade was meer afhankelijk
van de morfologie van deze osteofyten (i.e., een osteofyt die tot in het oppervlak van
een transplantaat groeit) dan van het percentage verkalking van het transplantaat.

In het tweede deel van dit proefschrift onderzochten we hoe het EO-proces beïnvloed
kan worden. In Hoofdstuk 4 toonden we aan dat cyclo-oxygenase-2 (COX-2) inhibitie

Addendum

niet alleen EO in de groeiplaat en in de fractuurcallus verminderde, maar ook de
periostale kraakbeenvorming in een in vivo konijnenmodel. De inhibitie van COX-2 werd
bereikt door het gebruik van celecoxib, een selectief COX-2 remmend medicijn dat valt
in de groep Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). Ondersteund door
voorgaande studies van onze onderzoeksgroep die een bifasisch patroon van COX-2
expressie in EO aantoonden, laten deze resultaten zien dat celecoxib niet alleen
hypertrofe differentiatie van kraakbeencellen remt, maar ook de chondrogene fase van
EO in bepaalde omstandigheden kan remmen.
Een andere manier om het EO-proces te beïnvloeden werd onderzocht in Hoofdstuk 5.
Het vervaardigen van kraakbeen uit progenitorcellen wordt vaak belemmerd door
ongewenste EO. Om het probleem van nadelige hypertrofe differentiatie van
chondrocyten tijdens ectopische kraakbeenvorming te voorkomen werd een eerder
gebruikte biogel aangevuld met de belangrijke kraakbeeneiwitten aggrecan of cartilage
oligomeric matrix protein (COMP). Deze eiwitten bleken in staat te zijn om het proces
van ectopische kraakbeenvorming te verbeteren in een in vivo konijnenmodel, door het
onderdrukken van hypertrofe differentiatie van het gevormde kraakbeenweefsel.

In het laatste deel van dit proefschrift werden potentiele behandelopties onderzocht
die het intra-articulaire milieu kunnen verbeteren om gewrichtspijn te verminderen en
progressie van kraakbeenschade naar artrose uit te stellen of te voorkomen. De huidige
beschikbare orale medicamenteuze behandelingen hebben vele nadelen die kunnen
worden overwonnen door intra-articulaire toediening van geschikte medicatie. Het
medicijn blijft echter vaak maar een korte tijd in het gewricht aanwezig en er is een
medicijnafgiftesysteem nodig om de effectiviteit van intra-articulaire therapie voor
kraakbeenschade te verbeteren. Verschillende soorten medicijnafgiftesystemen zijn:
Verlengde afgifte systemen, gecontroleerde afgifte systemen of autoregulatoire
systemen. De literatuurstudie verricht in Hoofdstuk 6 liet zien dat medicijn-
afgiftesystemen gemaakt kunnen worden van verschillende materialen. Sinds het
gebruik van polymeren voor de ontwikkeling van medicijnafgiftesystemen is er veel
progressie geboekt. Het gebruik van medicijnafgiftesystemen voor de behandeling van
artrose is veelbelovend, maar er is nog geen klinische trial die aangetoond heeft dat
deze medicijnafgiftesystemen in combinatie met medicijnen in staat zijn het
ziekteproces van artrose te veranderen. Hoofdstuk 7 van dit proefschrift beschrijft de
ontwikkeling en het testen van de polyester amide-celecoxib (PEA-CXB) micropartikel
als medicijnafgiftesysteem. De farmacokinetische eigenschappen en reactie op een
inflammatoire (artrotische) omgeving werden in vitro onderzocht in cellysaten
verkregen van een neutrofiele Hl-60 cellijn. Vervolgens werden de bio-compatibiliteit
en degradatie van de PEA-CXB micropartikel getest in een in vivo rat model waarbij
post-traumatische artrose was geïnduceerd door middel van het doornemen van de
voorste kruisband en gedeeltelijke verwijdering van de mediale meniscus. De PEA-CXB
micropartikels veroorzaakten geen detecteerbare bijwerkingen en waren geschikt als
medicijnafgiftesysteem. Een verandering van het artroseproces door de PEA-CXB
micropartikels werd niet aangetoond. In rattenknieën met artrose was er een

toegenomen degradatie van de micropartikels en de toevoeging van celecoxib aan de
partikels zorgde voor een afname van dezelfde degradatie, wat toebedeeld werd aan
auto-regulatoire eigenschappen van het medicijnafgiftesysteem. In Hoofdstuk 8
werden de belangrijkste bevindingen van dit proefschrift in breder perspectief
geplaatst.

De wereldwijde ziektelast van artrose stijgt snel, met name in relatief jonge patiënten.
Wanneer een totale knieprothese wordt geplaatst bij jonge patiënten is het risico op
een revisie operatie groot. Daarom is er meer aandacht nodig voor gewrichtssparende
behandelingen. Voor een succesvolle gewrichtssparende behandeling zijn een tijdige
herkenning, patiëntselectie en behandeling van kraakbeenschade en artrose nodig. Een
adequate behandeling kan bestaan uit leefstijl, farmacologische en/of chirurgische
interventies. De bevindingen in dit proefschrift tonen dat de verbetering van
gewrichtssparende behandelingen bereikt kunnen worden op verschillende niveaus en
vanuit verschillende perspectieven (bijvoorbeeld vanuit de cel en vanuit de patiënt).
Echter, het belangrijkste voor een succesvolle gewrichtssparende behandeling van
patiënten met gewrichtsschade is een verandering in de benadering van het klinische
probleem door orthopedisch chirurgen, andere zorgverleners en wetenschappers. Een
benadering waarbij de “patient journey” van een persoon met een gezond gewricht tot
aan een gewrichtsvervanging gevolgd en begeleid kan worden door middel van de
juiste interdisciplinaire samenwerking. Hierdoor wordt de juiste zorg op de juiste tijd en
plaats geboden om mensen en patiënten in beweging te houden en zo andere
chronische ziekten te voorkomen.

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Addendum

Impact paragraph

Impact paragraph

Cartilage is a durable, but flexible tissue that occurs throughout the body. In articular
joints, hyaline cartilage comprises a layer that covers the ends of the bones and
provides a surface with very low friction that makes movement possible and at the
same time functions as a shock absorber. Unfortunately, cartilage has a very low
healing capacity. Therefore, damage to articular cartilage is not resolved and often
leads to a pathway of joint deterioration and finally osteoarthritis (OA). Articular
cartilage lesions are found in up to 62% of the knees of adults without any symptoms of

joint pathology. When cartilage degradation becomes symptomatic, or even
progresses into OA, this can have an enormous impact on a person’s life. Osteoarthritis
is a leading cause of disability worldwide and its burden is only expected to increase
due to the ageing population and increasing incidence of obesity. Furthermore,
2,3
disabling OA leads to a substantially reduced long-term work participation and is
therefore a major economic concern as well. The most frequently applied therapy for

end-stage OA is arthroplasty, but the results of total knee arthroplasty (TKA) in working

patients are dissatisfying and one third of patients does not return to work after TKA.
The lifetime risk of implant revision is increased in younger patients (up to 35% for men
in their early 50s). In addition, the median time to revision is significantly shorter in

patients who were younger than 60 at the time of TKA. It is thus of great social and
economic value to prevent, or at least postpone progression towards end-stage OA and
subsequent (early) TKA. Therefore, the main goal of this thesis was to elucidate how
the process of endochondral ossification (EO) can be influenced to improve the
treatment of damaged cartilage (i.e., focal cartilage defects and OA).

Conclusion of main findings

The process of EO is an essential factor in cartilage damage and repair. The findings in
this thesis confirm that patient characteristics can negatively influence the outcome of
cartilage repair surgery. Potentially by impairing the joint homeostasis and increasing
joint inflammation and subsequent EO of the repaired cartilage tissue. The work in this
thesis underlines the potential of inhibiting inflammation and influencing the EO
pathway with the aim to improve cartilage repair and OA treatment by reducing
undesired chondrocyte hypertrophy.

Implications for research A

The influence of inflammation and patient characteristics on the outcome of cartilage
7,8
repair surgery and OA treatment is widely recognized, but still not fully understood.
The data in this thesis demonstrate that adequate patient selection can improve the
outcome of cartilage repair surgery. In addition, the added value of 7T MRI is
underlined. The detailed visualization of morphological and biochemical differences
(such as increased calcification of repaired cartilage) suggests that inflammation and EO

Addendum

can influence the results of articular cartilage repair. This shows that innovations in
imaging, such as (high-field) MRI, can aid in an increased understanding of the
mechanisms of treatment failure and subsequently provide directions to improve
treatment strategies. However, the influence of EO on the quality of cartilage repair
tissue and subsequent clinical outcome should be further elucidated in prospectively
designed studies. Increased knowledge on the use of 7T MRI also provides a way to
evaluate articular cartilage non-invasively and at multiple timepoints, facilitating future
clinical research on the influence of EO on articular cartilage damage and repair. This
future research could elaborate on the work presented in this thesis that describes the
potential improvement of ectopic cartilage tissue formation by influencing
inflammation and EO via the inhibition of cyclooxygenase (COX)-2. In addition, the
chondrocyte hypertrophy-suppressive effect of aggrecan and cartilage oligomeric
protein (COMP) without impairing cartilage formation provides an interesting starting
point for future studies.

Implications for individual patients and society
Good surgeons know how to operate, better ones when to operate, and the best when

not to operate. This was stated in a BMJ editorial dating back to 1999, but is still
applicable. Not performing unnecessary surgical procedures protects patients from
avoidable strain. In addition, it decreases hospital costs and all other socioeconomic
costs involved with the surgery. Key findings in this thesis increased the knowledge on
risk factors and adequate diagnostic tools to detect cartilage defects and (early) OA.
Early detection of cartilage damage provides the opportunity to improve the ‘patient
journey’ by starting early with a suitable treatment, preserve a functional joint and
prevent loss of mobility in patients. This can subsequently avoid costly procedures in
progressed OA such as revision of total knee arthroplasty or socioeconomic costs
caused by disability in patients of working age. Furthermore, an increased
understanding was obtained on the role of inflammation and EO on the treatment of
cartilage damage and OA by the development of the PEA-CXB microsphere. Derived
strategies could further elaborate on the inhibition of chondrocyte hypertrophy and
inflammation to treat cartilage damage and potentially lead to a reduction of the
amount of (early) TKA and subsequent revision TKA. Next to reduced socioeconomic
costs, a reduction in the amount of (early) TKA can also decrease secondary
(psychological) complaints and improve the quality of life of OA patients.

Implications for health care professionals

Next to the implications for the individual patient and society described above, the
research results presented in this thesis are valuable for health care professionals as
well. We found that late and multiple surgeries in older patients decrease the chance of
success in focal cartilage repair surgery. This underlines the importance of adequate
early treatment of articular cartilage damage to prevent further deterioration of the

Impact paragraph

joint. This can be achieved by educating primary physicians to recognize patients with
possible articular cartilage damage that are suitable for early referral to an orthopaedic
surgeon. This might be even more applicable for physical therapists, as in the Dutch
health care system, physical therapy is often the first line of treatment for patients with
(minor) musculoskeletal complaints. Orthopaedic surgeons can benefit from a timely
referral and potentially provide less invasive, joint preserving treatments.

Communication towards health care professionals
A timely treatment of articular cartilage damage can prevent further deterioration of
the joint. 10,11 In addition, the findings in this thesis showed that a timely treatment of
cartilage damage decreases the risk of treatment failure and subsequent TKA. To
facilitate this timely treatment of articular cartilage damage, primary healthcare
providers involved in the treatment of patients with focal cartilage defects or (early) OA
have to be taught that early referral can be joint-preserving. To educate health care
providers, publishing research results in peer reviewed journals is essential, but is not
enough. Medical information is abundantly available on the internet. However, keeping
an overview is complex and the abundant information is impossible to interpret and

apply for all different health care providers. For the results of this thesis (and other
research) to consistently reach all relevant health care providers, communication will
have to be improved. In the following years there will have to be significant
advancements in the infrastructure of electronic health records. The currently, not
directly linked electronic health records of (amongst others) primary physicians,
physical therapists and orthopaedic surgeons will have to be linked or integrated so
that all health care providers can have the access to relevant information and are
provided with adequate feedback on their treatment actions, ideally supported by
scientific research. This can be facilitated by the use of a personal health environment
in which personal medical data is owned by the patient and can be shared with
different institutions.

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Addendum