Clin Oral Invest
DOI 10.1007/s00784-017-2208-x
ORIGINAL ARTICLE
A 5-year prospective study on regenerative periodontal therapy
of infrabony defects using minimally invasive surgery
and a collagen-enriched bovine-derived xenograft
Thomas De Bruyckere 1,2 & Aryan Eghbali 1 & Faris Younes 1 & Roberto Cleymaet 1 &
Wolfgang Jacquet 1,2 & Hugo De Bruyn 2 & Jan Cosyn 1,2
Received: 1 February 2017 / Accepted: 11 September 2017
# Springer-Verlag GmbH Germany 2017
Abstract
Aim The primary objective of this study was (1) to evaluate
the 5-year clinical outcome of regenerative periodontal thera-
py (RPT) using minimally invasive surgery and a collagen-
enriched bovine-derived xenograft and (2) to identify predic-
tors for clinical attachment level (CAL) gain and vertical ra-
diographic bone (RB) gain.
Materials and methods Ninety-five non-smoking patients
with ≤ 25% full-mouth plaque and bleeding presenting
≥ 6 months after initial periodontal therapy with ≥ 1 isolated
interdental infrabony defect were recruited. Minimally inva-
sive surgery (MIST or M-MIST) and a collagen-enriched bo-
vine-derived xenograft were used in all patients. Patients were
surgically treated by the same clinician and evaluated up to
5 years of follow-up. Multivariate analyses were used to iden-
tify predictors for CAL gain and RB gain.
Results Before surgery, mean probing depth (PD) was 7.8 mm,
CAL was 10.0 mm, and defect depth amounted to 5.2 mm.
Seventy-one patients (33 men, 38 women, mean age 52) could
be evaluated at 5 years. Mean PD reduction was 3.3 mm (SD
2.2), CAL gain was 3.0 mm (SD 2.1), and RB gain was 57%
(SD 38). Forty-five percent showed ≥ 4 mm CAL gain, whereas
24% were considered failures (≤ 1 mm CAL gain). Forty-eight
percent showed considerable RB gain (≥ 75%). Regression
* Thomas De Bruyckere
thomas.de.bruyckere@vub.ac.be
1
Vrije Universiteit Brussel (VUB), Faculty of Medicine and
Pharmacy, Oral Health Research Group (ORHE), Laarbeeklaan 103,
B-1090 Brussels, Belgium
2
Faculty of Medicine and Health Sciences, Dental School,
Department of Periodontology and Oral Implantology, Ghent
University, De Pintelaan 185, B-9000 Ghent, Belgium
analyses showed that plaque was a significant predictor for
CAL gain (p = 0.001) and RB gain (p = 0.005). Patients’ com-
pliance had a significant impact on RB gain (p < 0.001).
Conclusion Only patients with perfect oral hygiene and ex-
cellent compliance should be considered for RPT. Especially,
the latter can only be assessed after sufficient follow-up fol-
lowing initial periodontal therapy.
Clinical relevance RPT failed in 24% of the patients after
5 years. Regression analyses demonstrated a significant im-
pact of plaque and patients’ compliance on the long-term out-
come. Only patients with perfect oral hygiene and excellent
compliance should be considered for RPT. Patients should not
be treated too soon following initial therapy, since compliance
can only be reliably assessed after sufficient follow-up.
Keywords Infrabony defect . Periodontal disease .
Regeneration . Xenograft . Minimally invasive surgery
Introduction
Infrabony defects have been identified as a risk factor for tooth
loss in the long term [1]. Therefore, their management has
always been a priority in periodontal care. Regenerative peri-
odontal therapy (RPT) has been proposed as a viable treatment
concept for these lesions with favorable clinical and radio-
graphic outcomes [2].
Various flap designs have been described for RPT. Takei
et al. [3] introduced the so-called Bpapilla preservation flap^ in
1985, which was later modified and simplified for wide and
small interdental spaces, respectively [4, 5]. Also, the so-
called Bsingle flap approach^ was introduced with a clear
focus on optimal soft tissue preservation [6]. These flap de-
signs were clinically implemented in the minimally invasive
surgical technique (MIST) [7], or a modification of that

technique (M-MIST) [8]. Both are based on soft tissue pres-
ervation and tension-free primary closure contributing to a
stable wound and space maintenance for cellular ingrowth.
In clinical practice, periodontal regeneration is usually fa-
cilitated by means of biomaterials. In this field, mainly three
groups can be distinguished: barrier membranes used for guid-
ed tissue regeneration (GTR), bone replacement grafts (allo-
grafts, xenografts, alloplastic materials), and wound modifiers
(enamel matrix derivative (EMD)). Also, combinations have
been used and clinically evaluated [9].
The ultimate goal of RPT is to achieve regeneration of
alveolar bone, root cementum, and periodontal ligament
[10]. Histological studies have demonstrated such regenera-
tion for GTR [11–13], allografts (Bowers et al. 1989), xeno-
grafts [14–21], and enamel matrix derivative (EMD) [21–23].
Long-term studies demonstrated stable clinical results up to
10 years and even longer [24–28].
A prerequisite for broad application of RPT is procedural
simplicity. RPT by means of EMD may be considered straight-
forward in this respect in contrast to GTR, which requires high-
ly skilled surgeons for meticulous membrane positioning. In
addition, significantly more complications may be expected fol-
lowing GTR when compared with EMD application [29]. On
the other hand, EMD may not be ideal for treating wide defects
[30], especially those with a non-supportive anatomy [31]. The
use of a collagen-enriched bovine-derived xenograft may over-
come some of these limitations. Clinical and histological studies
have demonstrated periodontal regeneration following the ap-
plication of such a graft in infrabony defects with and without a
membrane [15–20, 32]. To the best of our knowledge, there are
no long-term studies on the clinical and radiographic outcome
of RPT using minimally invasive surgery and a collagen-
enriched bovine-derived xenograft. Hence, the primary objec-
tive of this study was to evaluate the 5-year outcome of this
treatment concept in a large cohort. The secondary objective
was to identify predictors for clinical attachment level (CAL)
gain and vertical radiographic bone (RB) gain in the long term.
Materials and methods
Patient selection
Ninety-five patients were consecutively treated between
March 2008 and January 2011 for at least one infrabony defect
by the same clinician (JC). Patients were treated in two private
practices and at the University Hospital in Ghent. They were
selected during a screening visit on the basis of inclusion and
exclusion criteria.
Inclusion criteria were as follows:
& Good oral hygiene defined a full-mouth plaque and bleed-
ing score ≤ 25% [33]
Clin Oral Invest
& Initial periodontal therapy (scaling and root planning and
oral hygiene instructions) terminated at least 6 months pri-
or to screening
& Presence of at least one isolated interdental infrabony de-
fect around a single-rooted tooth or mandibular molar (flat
surface) with ≥ 3 mm radiographic depth and ≥ 6 mm
probing depth
& Signed informed consent
Exclusion criteria were as follows:
& Systemic diseases
& Smoking
& History of surgical treatment at the tooth with the
infrabony defect
& Adjacent teeth with infrabony defects
& Furcation involvement
The study was conducted in accordance with the Helsinki
declaration of 1975 as revised in 2000 and the study protocol
was approved by the ethical committee of the University
Hospital in Ghent.
Surgical approach
A minimally invasive surgical technique (MIST) or modifica-
tion (M-MIST) was used [7, 8]. Surgery was preceded by oral
and peri-oral disinfection using chlorhexidine 0.2%. A beveled
incision was performed with a microsurgical blade (stainless
fine steel surgical blade Swann-Morton, Sheffield, England)
and was limited to the interdental area with the infrabony de-
fect, if possible. If access to the bottom of the defect was
deemed insufficient, the flap was extended to the neighboring
interdental area. A simplified papilla preservation flap [5] was
performed whenever the interdental space was 2 mm. In case of
a wide inter dental space, a modified papilla preservation flap
[4] was performed. Whenever the infrabony defect did not ex-
tend to the palatal/lingual side, the modified minimally invasive
surgical technique (M-MIST) was performed. That is, flap ele-
vation was limited to the buccal side, hereby keeping the
supracrestal interdental tissues attached to the palatal/lingual
side. Upon flap reflection, granulation tissue was dissected
and root debridement was performed using ultrasonic and hand
instruments. Chemical root conditioning was never performed.
Following intra-surgical data registration, a collagen-enriched
bovine-derived xenograft (Bio-Oss Collagen® 100 mg;
Geistlich Biomaterials, Wolhusen, Switzerland) was individu-
alized, applied into the defect, and slightly condensed.
Attention was paid not to overfill the defect. Finally, the flap
was replaced at the original position. Tension-free primary clo-
sure of the interdental papilla was attained by means of an
internal vertical matrass suture using monofilament material
(Seralon® 5/0; Serag Wiessner, Nail, Germany). Neighboring
Clin Oral Invest
interdental areas were closed by means of a single suture.
Figure 1 illustrates the surgical procedure. Postoperative med-
ication included amoxicillin 1000 mg (twice a day during
4 days), ibuprofen 600 mg (as deemed necessary by the
patient), and local application of chlorhexidine 0.2% by
means of a spray (twice a day during 2 weeks). Mobile
teeth were splinted and interdental cleaning was omitted
during 1 month. After 2 weeks, sutures were removed and
teeth were polished using chlorhexidine gel (1%). The
latter was repeated after 1 month. Patients received sup-
portive periodontal treatment every 3 months; after 1 year
follow-up, recall intervals were tailored to the needs of
the patient. Supportive periodontal therapy included pro-
fessional plaque and calculus removal and polishing. If
necessary, oral hygiene was reinforced.
Outcome variables
The following clinical/radiographic parameters were regis-
tered by the same clinician (JC):
1. Plaque score was registered at the deepest interdental
point of the defect before surgery, after 1 month, 3 months,
1 year, and 5 years. A dichotomous score was given
(0 = no visible plaque at the gingival margin; 1 = visible
plaque at the gingival margin).
2. Probing depth (PD) was registered at the deepest inter-
dental point of the defect to the nearest 0.5 mm using a
manual probe (CP 15 UNC; Hu-Friedy®, Chicago,
USA). Probing depth was measured before surgery, after
1 year, and after 5 years.
3. Recession (REC) was registered at the deepest interdental
point of the defect (interdental REC) and at the midfacial
Fig. 1 Surgical procedure. a
Modified minimally invasive
surgical technique. b
Predominantly two-wall defect of
5 mm depth. c Defect filled with
collagen-enriched bovine-derived
xenograft. d Modified internal
vertical matrass suture at the
mesial aspect of tooth 45 and
single suture at the distal aspect
aspect (midfacial REC) to the nearest 0.5 mm using the
same manual probe. REC was defined as the distance
from the free gingival margin to the cement-enamel junc-
tion and was measured before surgery, after 1 year, and
after5 years.
4. Patients’ compliance. A dichotomous score was given
(1 = patient attended the visits at 1 month, 3 months,
1 year, and 5 years; 0 = patient missed one of these visits).
5. Clinical attachment level (CAL) was calculated for the
deepest interdental point of the defect as the sum of PD
and REC before surgery, after 1 year and 5 years.
6. Vertical radiographic bone (RB) gain was calculated as
the difference between the defect depth (distance in mm
between bottom of the defect and the alveolar crest) be-
fore surgery, after 1 year, and after 5 years. The parameter
was expressed as a percentage of the original defect depth.
Data were based on digital intra-oral radiographs taken
before surgery, after 1 year, and after 5 years, using the
paralleling technique. Designated software was used for
linear measurements (DBSWIN; Dürr Dental AG,
Bietigheim- Bissingen, Germany) (Fig. 2).
The following intra-surgical parameters were registered by
the same clinician (JC):
1. Defect anatomy. A distinction was made between pre-
dominantly one-wall, predominantly two-wall, and pre-
dominantly three-wall infrabony defects.
2. Defect depth was defined as the distance in millimeters
between bottom of the defect, and the alveolar crest was
registered at the deepest interdental point of the defect.
The parameter was measured to the nearest 0.5 mm using
the same manual probe.
 Clin Oral Invest

Fig. 2 a Pre-operative radio-

graph. Defect depth: 5 mm.
b Radiograph after 5 years.
Vertical radiographic bone gain,
100%

Statistical analysis could not be traced anymore. The remaining 71 patients were 33
men and 38 women with a mean age of 52 years.
The patient was the statistical unit in all analyses. Data anal-
ysis was performed using the statistical software package
Clinical outcome
SPSS 22 (SPSS Inc., Chicago, IL, USA). If more than one
isolated interdental infrabony defect was treated in the same
The 1-year clinical outcome of the present cohort was pub-
patient, the defect closest to the midline was selected [34].
lished earlier [34]. At 5 years, mean PD reduction was 3.3 mm
Data analysis included descriptive statistics with frequency
(SD 2.2; range 0.0–8.0), mean CAL gain 3.0 mm (SD 2.1;
distributions on PD, REC, CAL gain (treatment failure
range 0.0–0.7), and mean RB gain amounted to 57% (SD 38;
≤ 1 mm CAL gain; 2–3 mm CAL gain; ≤ 4 mm CAL gain),
range 0–100). Twenty-four percent demonstrated 0–1 mm
and RB gain. Paired comparisons for PD, REC, CAL gain,
CAL gain and were considered failures, 31% showed 2–
and RB gain between 1- and 5-year registrations were per-
3 mm CAL gain, and 45% showed ≥ 4 mm CAL gain.
formed by means of the paired samples t test. A general linear
Forty-eight percent showed considerable RB gain (≥ 75%).
model was used to examine the association of defect depth,
Table 1 shows the paired comparisons for PD, REC, CAL
number of bone walls (1, 2, or 3), surgical procedure, patients’
gain, and RB gain between 1 and 5 years. There were no
compliance, and plaque score with CAL gain and RB gain
significant differences in any of these parameters within this
(dependent variables). A residual analysis on linearity and
time frame (p ≥ 0.171).
homoscedasticity was performed to evaluate the model of
fit. The level of significance was set at 0.05.
Predictors for CAL gain and RB gain

Table 2 shows the results of the regression analysis with CAL

Results
Patient selection
Eighty-four patients (39 men, 45 women; mean age 53) complied
up to 1 year and demonstrated mean PD of 7.8 mm (SD 1.5;
range 6.0–12.0), CAL of 10.0 mm (SD 2.3; range 6.0–17.0), and
radiographic defect depth of 5.2 mm (SD 1.7; range 3.0–10.0)
before surgery. Between 1 and 5 years, there were 13 additional
drop outs. Patients were unwilling to return for reassessment or
gain as the dependent variable. The data showed no
multicollinearity. Plaque was the only significant predictor
for CAL gain (p = 0.001). In fact, presence of plaque on at
least 1 reassessment resulted in 1.6 mm CAL loss according to
the regression model. Fifty-seven percent of the variability
could be explained by the regression model (R2 = 0.572).
The model quality was satisfying given the linear relationship
and homoscedasticity of the residuals (Fig. 3, left side). An
illustration on the prediction of CAL gain by the regression
model is given in Fig. 3 (right side).

Table 1 Paired samples t test

showing no statistically Comparison between 1- and 5-year results
significant difference in mean
pocket depth (PD) reduction, Mean Standard 95% Confidence interval p value
recession (REC), clinical deviation Lower Upper
attachment level (CAL) gain, and PDreduction1year–PDreduction5years − 0.20 mm 1.48 − 0.55 0.15 0.265
vertical radiographic bone (RB) REC1year–REC5years − 0.15 mm 0.90 − 0.36 0.07 0.171
gain between 1 and 5 years
CALgain1year–CALgain5years − 0.04 mm 1.60 − 0.41 0.34 0.853
RBgain1year–RBgain5years 2.03% 14.45 − 1.71 5.76 0.282

Negative value indicates deterioration of the outcome variable, whereas a positive value indicates improvement
Clin Oral Invest

Table 2 Regression analysis

with CAL gain (mm) as Regression analysis with CAL gain as dependent variable
dependent variable. R2 = 0.572
Parameter Regression Standard error 95% Confidence p value
coefficient interval
Lower Upper
Intercept 4.02 0.87 2.32 5.73 < 0.001
Defect depth 0.04 0.15 − 0.26 0.33 0.807
Number of bone walls 1 vs. 3 − 0.81 0.76 − 2.31 0.68 0.285
2 vs. 3 0.21 0.58 − 0.92 1.34 0.711
Surgical procedure (MIST vs. − 0.41 0.46 − 1.32 0.50 0.375
M-MIST)
Patients’ compliance (1 vs. 0) 0.49 0.53 0.55 1.54 0.356
Presence of plaque on at least 1 − 1.55 0.48 − 2.49 − 0.62 0.001
reassessment (1 vs. 0)

A similar regression analysis was performed with RB gain
as dependent variable (Table 3). The data showed no
multicollinearity. Predictors for RB gain included plaque
(p = 0.005) and patients’ compliance (p < 0.001). In fact,
presence of plaque on at least 1 reassessment resulted in
23% RB loss, whereas full compliance increased RB gain by
37% according to the regression model. Sixty-eight percent of
the variability could be explained by the regression model
(R2 = 0.676). The model quality was also satisfying given
the linear relationship and homoscedasticity of the residuals
(Fig. 4, left side). An illustration on the prediction of RB gain
by the regression model is given in Fig. 4 (right side).
Discussion
In the present prospective study, the 5-year clinical outcome of
RPT using minimally invasive surgery and a collagen-
enriched bovine-derived xenograft was evaluated. To the best
of our knowledge, there are no other long-term studies on this
treatment concept.
Comparing the 1- and 5-year clinical results, stable clinical
conditions could be shown. In addition, mean CAL gain of
3.0 mm seems to correspond with other long-term studies on
the clinical outcome of GTR [26–28], RPT using EMD [27],
or RPT using a combination of EMD and GTR [27] with mean
CAL gains ranging from 2.4 to 3.8 mm. Mean values do not
provide information on the prevalence of success or failure. In
that respect, frequency distributions may be more informative.
These findings suggest a slight increase in failure rate from 1
to 5 years (15 to 24%). This evolution may reflect a true
deterioration that is masked when evaluating data in terms of
mean values. Alternatively, it could be the result of patient
dropout amounting to 25% at 5 years. Even though a failure
rate of 24% may be considered quite high, regression analyses
indicated superior outcomes in highly compliant and plaque-
free patients. Indeed, presence of plaque on at least one reas-
sessment resulted in 1.6 mm CAL loss after 5 years. Likewise,
presence of plaque on at least 1 reassessment resulted in 23%

3,00 8,00
Standardized Residual for CAL gain

Predicted Value for CAL gain (mm)

2,00 6,00

1,00 4,00

,00 2,00

-1,00 ,00

-2,00 -2,00

-2,00 ,00 2,00 4,00 6,00 8,00 -2,5 ,0 2,5 5,0 7,5

Predicted Value for CAL gain (mm) CAL gain (mm)

Fig. 3 Scatter plot illustrating the model quality in terms of linearity and homoscedasticity (left side). Scatter plot illustrating the prediction of CAL gain
by the regression model (right side)
 Clin Oral Invest

Table 3 Regression analysis

with RB gain (%) as dependent Regression analysis with RB gain as dependent variable
variable. R2 = 0.676
Parameter Regression Standard error 95% Confidence p value
coefficient interval
Lower Upper
Intercept 104.92 15.10 75.32 134.52 < 0.001
Defect depth − 2.28 2.39 − 6.96 2.41 0.341
Number of bone walls 1 vs. 3 − 15.40 13.07 − 41.02 10.21 0.239
2 vs. 3 − 2.62 10.04 − 22.30 17.07 0.794
Surgical procedure (MIST vs. − 13.53 7.82 − 28.86 1.80 0.084
M-MIST)
Patients’ compliance (1 vs. 0) 37.30 9.35 55.62 18.98 < 0.001
Presence of plaque on at least 1 − 23.24 8.25 − 39.40 − 7.08 0.005
reassessment (1 vs. 0)

RB loss, whereas full compliance increased RB gain by 37%
after 5 years. The clinical implication of these findings is that
the predictability of RPT can be improved by very stringent
case selection. Only patients with perfect oral hygiene and
excellent compliance should be considered. Especially, the
latter can only be assessed after sufficient follow-up following
initial periodontal therapy.
In this study, 47% of the patients demonstrated perfect oral
hygiene as they were plaque-free at the surgical site during all
assessments (before surgery, 1 month, 3 months, 1 year, and
5 years). Sixty-three percent of the patients were fully compli-
ant, meaning attending all visits. The latter is in agreement
with the available literature on compliance of patients follow-
ing periodontal therapy. Roughly 50% of the patients may be
expected to be fully compliant, one third is fairly compliant,
and a minority does not show up anymore during aftercare
(43, 44). In this context of this study, no data were registered
between 1 and 5 years of follow-up. This does not imply that
patients were not evaluated in this time period. All patients
received supportive periodontal therapy with an interval
tailored to patients’ needs. These intervals varied between 3,
6, and 12 months.
Studies have shown that an optimal surgical approach may
outweigh the need for biomaterials [35, 36]. Indeed, open flap
debridement may result in mean CAL gain up to 3.5 mm or
more when using a minimally invasive approach, which
seems in line with our findings and those of multi-center stud-
ies on RPT [31, 37–39]. These findings do not suggest that
biomaterials are redundant, but that cases should be carefully
selected for a cost-effective application of these materials.
Especially, deep and wide infrabony defects without support-
ive anatomy may benefit from their use [40, 41].
Given the doubtful or even poor initial prognosis of the
treated teeth (mean CAL 10.0 mm), some may consider a sin-
gle implant a viable alternative for RPT. The fact that a failure
rate of 24% was found for RPT in this study could be an extra
argument for non-believers of RPT. On the other hand, a num-
ber of aspects could also favor RPT. First, success and failure
are defined in different ways in oral implant dentistry and peri-
odontology. An implant is usually considered a failure when it

2,00 100,00
Standardized Residual for RB gain

Predicted Value for RB gain (%)

80,00
1,00

60,00
,00

40,00

-1,00
20,00

-2,00
,00

-3,00 -20,00

-20,00 ,00 20,00 40,00 60,00 80,00 100,00 0 20 40 60 80 100

Predicted Value for RB gain (%) RB gain (%)

Fig. 4 Scatter plot illustrating the model quality in terms of linearity and homoscedasticity (left side). Scatter plot illustrating the prediction of RB gain
by the regression model (right side)
Clin Oral Invest
is lost, whereas RPT is considered a failure in case of CAL loss
or minute CAL gain, usually with the tooth still in situ. Clearly,
such a definition is much more conservative. Second, implant
treatment is generally more expensive than RPT and in case of
failure of the latter an implant remains an option. Finally, RPT
can be successful for severely compromised teeth and has the
potential to change their prognosis, be it under strict conditions
[42]. These include careful patient selection, an appropriate
surgical technique, and a skilled surgeon.
When interpreting the results of this study, several limita-
tions should be acknowledged. First, this is not a RCT. Given
the objectives, however, it was not our intention to compare
treatment modalities. Second, all patients had been treated by
one clinician, which could have an impact on the external
validity of the results of this study. Third, patients were treated
and evaluated by the same clinician, which could impose bias.
This is related to the fact that this study was mainly run in a
private setting where it was not possible because of logistics to
have patients evaluated by another investigator. Finally, 24
dropouts were registered after 5 years. Ample studies confirm
that many patients do not adhere to supportive periodontal
care programs [43, 44].
Conclusion
In conclusion, the present prospective study included data on
71 patients who were treated with RPT and could be followed
for 5 years. Mean CAL gain was 3.0 mm with nearly half of
the cases showing ≥ 4 mm CAL gain. However, 24% of the
cases were considered failures (CAL gain ≤ 1 mm). Oral hy-
giene and patients’ compliance had a pivotal impact on the
long-term outcome as shown by regression analyses.
Therefore, only patients with perfect oral hygiene and excel-
lent compliance should be considered for RPT. Patients should
not be treated too soon following initial therapy, since compli-
ance can only be reliably assessed after sufficient follow-up.
Compliance with ethical standards The study was conducted in ac-
cordance with the Helsinki declaration of 1975 as revised in 2000 and the
study protocol was approved by the ethical committee of the University
Hospital in Ghent.
Conflict of interest Author Drs. Thomas De Bruyckere declares that he
has no conflict of interest. Author Dr. Aryan Ehgbali declares that he has
no conflict of interest. Author Drs. Faris Younes declares that he has no
conflict of interest. Author Prof. Roberto Cleymaet declares that he has no
conflict of interest. Author Prof. Wolfgang Jacquet declares that he has no
conflict of interest. Author Prof. Hugo De Bruyn declares that he has no
conflict of interest. Prof. De Bruyn has a research and education collab-
oration agreement with Densply, Sirona, and Southern Implants (on be-
half of Ghent University). Author Prof. Jan Cosyn declares that he has no
conflict of interest. Prof. Cosyn has a collaboration agreement with Nobel
Biocare.
Funding The study was self-supported, but the company Mediplus,
Rixensart, Belgium, provided free materials to be used in the study.
Prof. Cosyn has a collaboration agreement with Mediplus, Rixensart,
Belgium.
Ethical approval All procedures performed in studies involving hu-
man participants were in accordance with the ethical standards of the
institutional research committee and with the 1964 Helsinki declaration
and its later amendments or comparable ethical standards.
Informed consent Informed consent was obtained from all individual
participants included in the study.
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