Peri-implantitis, implant loss and osteoradionecrosis in oral cancer patients

Each year, on average, 520 new cases of cancer of the oral cavity are diagnosed
in the Netherlands. This represents 0,9% of all newly diagnosed cases of
cancer. The treatment for oral cancer consists, depending on localisation and
staging, of surgery and/or radiotherapy(1). Oncologic resections in oral cancer
patients are often complex and mutilating. Reconstruction with
osteomyocutaneous flaps are commonly performed in these patients. This is a
major surgical procedure that has extensive consequences for oral functions
such as swallowing, chewing and speech. Many patients need oral rehabilitation
after the treatment for oral cancer, which consists of logopedia and dental
prosthetics.

Oral rehabilitation with dental implants of patients treated for oral cancer is
a meaningful postoperative procedure to increase quality of life(2). Dental
implant surgery is often indicated in these patients to create a sound fitting
prosthesis in the altered anatomy of the reconstructed oral cavity. Though
radiotherapy plays an important role in the treatment of head and neck
malignancies, side effects occur that have a major impact on the final results
of oral rehabilitation. Irradiation of the oral cavity is known to give acute
and chronic adverse local effects such as mucositis, xerostomia, hypogeusia,
trismus, radiation caries, candidiasis and osteoradionecrosis(3).

Osteoradionecrosis (ORN) is a severe complication and is characterized by a
non-healing area of exposed bone of at least three months duration(4). ORN is
associated with pain, drainage and fisteling of the mucosa or skin and usually
has a high morbidity. In advanced stages, ORN typically requires surgical
resection and reconstruction. Its incidence in de mandible is between 2-22% of
irradiated oral cancer patients. Factors that are thought to affect the
development of ORN include site and size of tumour, dose of irradiation, type
of resection, trauma (dental extractions), infection, immune deficiencies and
malnutrition(5). Placement of dental implants increases the risk of ORN.
Placing dental implants in irradiated tissue therefore is a clinical challenge.
Implant loss is observed in up to 24% of these patients(6).

Little is known about the etiology of ORN. During the past 80 years a number of
theories have been proposed, with consequent implications for its treatment. In
1983, Marx proposed the hypoxic-hypocellular-hypovascular theory: after
irradiation hypoxic-hypocellular-hypovascular tissue is formed, and breakdown
of this tissue driven by persistent hypoxia can cause a chronic non-healing
wound(7). This theory formed the basis of the use of hyperbaric oxygen therapy
for the treatment of ORN. Conflicting opinions about its efficacy exists, which
is reflected in the literature(8).

In 2004 another hypothesis was proposed: that of the fibro-atrophic process. In
this model, three successive clinical and histopathological phases can be
distinguished: a pre-fibrotic aspecific inflammatory phase, a constitutive
fibrotic cellular phase, and a matrix densification and remodelling phase,
possibly ending in terminal tissular necrosis(9). In the fibrotic process
different cytokines and growth factors are thought to play a role, such as
TNF-*, PDGF, FGFb, IL1,4 and 5, TGF*-1 and CTGF. TGF-*1 has been associated
with different fibrotic processes such as atherosclerosis, kidney, liver and
lung fibrosis and is thought to play a key role in post-radiation fibrosis. The
exact cascade of events and role of different cytokines and growth factors in
this process has not yet been clarified.



In order to avoid or decrease irradiation-induced complications, it is of vital
importance to get more insight in the histological and molecular background of
the effects of irradiation on oral soft and hard tissues. Previous studies have
focused on the effects of irradiation on bone tissue. Radiogenic bone
damage(10-17) and osseo-integration of implants in irradiated bone(18-22) has
been studied in various animal models. In irradiated rat mandible models,
diminished bone volume and mineral apposition rate were observed al well as
diminished numbers of osteoclasts and osteoblasts, hypovascularity, presence of
fibrosis and inflammation and unusually high presence of osteoclasts in
extraction sockets10,11.With regards to osseointegration of implants in animal
models, a dose dependent relationship between radiotherapy and osseointegration
is observed: the osseointegration diminishes with increasing dose of
irradiation(18-22).

This relationship has also been described in a retrospective analysis of dental
implant survival after radiotherapy in humans(23). Although the data with
regard to irradiated bone are not conclusive, it has clearly been shown that
after irradiation, there is a higher chance of failure of dental
implants(6,23-24). However evidence for a dose-dependent relationship of
irradiation dosage and implant failure in humans is conflicting. One systematic
review on dental implant osseointegration after radiotherapy suggests that
increased implant loss occurs in patients that received a radiation dose that
exceeds 50 Gray(24), whereas another describes no dose-dependent relationship
with regards to osseointegration(6). A possible contributing factor to these
conflicting reports is that radiotherapy went through a rapid development over
the past decades with respect to dosimetry and strategies to keep normal
anatomical structures outside the irradiation field(25-27). It has been
suggested that these more sophisticated methods of irradiation contribute to
lower incidence of ORN(27). However, improved dosimetry in radiotherapy regimes
has not been identified as an independent factor for reducing the risk of ORN
or implant loss in clinical setting with long-term follow-up.

In the current literature, no studies focusing primarily on histological and
histomorphometric changes in irradiated human jaw bone could be found. One
study has performed a histological and histomorphometrical evaluation of bone
tissue adjacent to post-mortem retrieved dental implants from irradiated head
and neck cancer patients(28). This study focused on osseointegration by
measuring bone-implant contact. In the retrieved dental implants, lowered
metabolic activity of bone tissue and diminished bone-implant contact was
observed. This finding suggests that irradiation has effect on bone metabolism
in human jaw bone that can possibly be objectified by performing histological
and histomorphometrical analysis of irradiated human jaw bone as proposed in
this research protocol.

One of the key processes of ORN is thought to be the altered expression of
inflammation mediators and growth factors. In order to assess altered
expression of genes in tissue a micro-array analysis can be performed(29). A
micro-array analysis detects levels of different strands of mRNA in a tissue
sample. When a gene is expressed, mRNA is transcribed from DNA and serves as a
template for protein synthesis. Because an mRNA transcript is an exact copy of
a corresponding DNA coding region, genomic analysis at the mRNA level can be
used as a measure of gene expression. Previous studies using microarray
analysis in oral cancer patients have mainly focused on gene expression of
tumor cells(30-33). Few studies focusing on the effect of irradiation on the
tissues surrounding the tumor could been found in the literature. In one study,
a microarray analysis of dermal keratinocytes in radiation induced skin wounds
demonstrated altered expression of keratins, growth factors and matrix
metalloproteinases(34). Another study demonstrated altered gene expression in
peripheral blood cells following adjuvant chemoradiation therapy for head and
neck cancer(35). Late changes in cutaneous gene expression after radiotherapy
for head and neck cancer were demonstrated in microarray analysis of
irradiated, non-ulcerating skin specimen harvested at secondary corrective
surgical procedure(36). However, expression profiles of oral tissues involved
in osteoradionecrosis have not yet been studied.

In the field of rheumatology, the microarray technique has been used to
identify expression patterns of inflammatory mediators in rheumatoid arthritis
patients, that could to some extent be correlated with clinical parameters such
as disease activity and biomarkers in peripheral blood(37,38). This finding
suggest that certain phenotypes of immune response can predispose for
pathologic inflammatory conditions such as auto-immune diseases. According to
the fibro-atrophic theory, ORN, like rheumatoid arthritis is considered a
pathologic inflammatory response. It is possible that a specific *fingerprint*
of inflammation response, as found in rheumatoid arthritis patients, can play a
part in the etiology of ORN. Therefore, it will be interesting to perform a
gene expression microarray analysis of irradiated human mucosa versus human
mucosa involved in ORN.

The effects of irradiation on mucosal cells can be observed in scanning
electron microscopy. Scanning electron microscopy (SEM) studies have shown that
the outer surface of epithelial cells of the oral mucosa is covered by numerous
microplicae (microvilli/ microprojections)39. Several functions have been
attributed to this characteristic structure. It is thought to maximize the
uptake of oxygen and nutrients and facilitate the movement of metabolic
products across the outer cell membrane. Microplicae functions are thought to
be closely related to glycocalyx and extracellular matrix (ECM) sturcture.37
Although the exact function of microplicae remains unclear, it is widely
accepted that this structure is essential to maintain intact tissue
physiology40. Scanning electron microscopy of irradiated buccal human mucosa
swaps have shown loss of microplication and microvilli of the mucosal
cells41,42, however, cells retrieved from buccal smears are subject to bias due
to contamination and lack of tissue structure. It would be a great value to
investigate the effects of irradiation on the mucosal tissue in humans by
performing scanning electron microscopy on mucosal tissue specimen surfaces as
opposed to buccal swaps, hereby creating an image that also contains the
intercellular junctions, maintaining normal tissue architecture and diminishing
contamination with mucus and bacteria.

Hyaluronan (HA) is a multifunctional glycosaminoglycan that forms the
structural basis of the extracellular matrix (ECM). Hyaluronan synthases (HASs)
are plasma membrane enzymes that simultaneously elongate, bind, and extrude the
growing hyaluronan chain directly into extracellular space. Hyaluronan (HA) is
thought to promote cancer cell growth and migration43. HA has been shown to be
a prognostic marker in oral squamous cell carcinoma, and a diminished intensity
of HA staining is associated with poor survival44. HASs play a role in wound
healing and in some oral diseases45. The role of hyaluronan (HA) in irradiated
oral tissues has not yet been researched. Altered expression of hyaluronan
could have structural effects on the extracellular matrix, that we assume does
not only play a role in invasive cancer growth, but also in the altered wound
healing in irradiated mucosa.

Our hypothesis is that peri-implantitis, implant failure and ORN in irradiated
oral cancer patient is caused by abnormalities in both hard and soft oral
tissues, possibly with similar molecular mechanisms. This study aims to
investigate the process of irradiation-induced changes in both jaw bone and
oral mucosa. By investigating gene expression of inflammatory markers,
histological properties and cell surface and extracellular matrix changes of
the irradiated versus non-irradiated oral tissues we aim to find clues to
predict future peri-implantitis, implant loss and ORN.

In the Department of Oral and Maxillofacial Surgery (OMFS) of the VU University
medical center in Amsterdam there is a longstanding and large experience with
the reconstruction and oral rehabilitation with dental implants in oral cancer
patients. During many years there has been a collaboration in the field of bone
research between the department of oral and maxillofacial surgery and the
department of endocrinology. There are strong bonds with the Finnish experts in
the field irradiated oral tissues, especially at electron microscopy level.


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Primary Objective:
What are the consequences of irradiation for the human jaw bone and oral mucosa
on a macroscopic, microscopic and molecular level?
Can possible prognostic factors be identified for the occurrence of
peri-implantitis, dental implant loss and/or ORN in irradiated patients with
oral cancer?
Is it possible to enhance the treatment decisions and prognoses of patients
with dental implants in reconstructive surgery?

Secondary Objective(s):
1. By performing histomorphometric analysis and micro-CT scanning of the
irradiated bone samples retrieving data on:
- Histological features of irradiated, osteoradionecrotic and 'control' bone *
Histology
- Number and size of blood vessels * Histology
- Number of osteoblasts per tissue area (N.Ob/T.Ar, cells/mm2), osteoclasts per
surface area (N.Oc/T.Ar, cells/mm2), osteocytes (N.Ot/Md.Ar. Cells/0,01 mm2)
and empty lacunae per mineralized surface area (N.Lac/Md.Ar. Cells/0,01 mm2) *
Histomorphometry
- Number of osteocytes that show signs of apoptosis (N.Apopt/Md.Ar. Cells/0,01
mm2) * Immunohistochemical staining of cleaved PARP
- Trabecular width (Tb.Wi., um) * Histomorphometry
- Trabecular thickness (Tb.Th. , um) * Micro CT
- Osteoid width (O.Wi., um) * Histomorphometry
- Osteoid thickness (O.Th, um) * Micro CT
- Absolute osteoid volume (OV/TV, %) * Micro CT
- Relative osteoid volume (OV/BV, %) * Micro CT
- Resorption surface - Histology
- Labeled surface (mineral apposition rate) (tertracycline labeling) *
Fluorescence Microscopy
- Surface area of bone, fibrosis, necrosis and marrow * Histology
- Presence of inflammatory cells/macrophages - Histology
- Calculating bone volume (BV), tissue volume (TV) and vital bone volume
(BV/TV, %) * Micro-CT
- Anisotropy (direction of trabecles) (Tr.Pf) * Micro CT
- Differences in types of irradiated bone (after surgery, depending on
reconstruction, the jaw bone can consist of original mandibular/maxillar, or
transplanted autograft (fibula, crista) bone) * Histology

2. By performing micro array analysis on irradiated mucosa, retrieving data on
- Gene-expression of proteins (focusing on cytokines and growth factors) in
post-cancer non-irradiated, post cancer irradiated and osteoradionecrotic
tissue compared to healthy smoking an non-smoking controls
- Determine what proteins are differently expressed in clinically evident
osteoradionecrosis
- Determine whether different expression profiles exist between irradiated
patients
- In case these different expression profiles are found, correlate these with
clinical data, investigating whether certain expression profiles in irradiated
tissue predispose for developing peri-implantitis, ORN and dental implant loss
- Significantly differently expressed inflammatory proteins, growth factors and
cytokines can be investigated on tissue level with immunohistochemistry
analysis to assess presence and localisation in the tissue.

3*. By performing scanning electron microscopy and different non-invasive
microspectrosopy on healthy, post-cancer, post-cancer irradiated and
post-cancer irradiated radionecrotic mucosa and bone tissue specimens,
obtaining information about:
- Cell surface changes in oral cancer mucosa, irradiated mucosa and
osteonecrotic mucosa compared to healthy controls
- Changes in microplication/microvilli and their interaction with hyaluronan
biosynthesis



* This part of the research will be performed at the SIB-labs Laboratory of the
University of Eastern Finland, Kuopio, Finland.

Prospective observational study

Venapuncture for blood sampling is an invasive procedure which may cause slight
discomfort for the patient.
Tetracycline labeling of bone has a potential risk for adverse reactions that
are known for tetracycline use and are described in the 'farmacotherapeutisch
kompas'.