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Received: 24 October 2017    Revised: 18 April 2018    Accepted: 13 May 2018

DOI: 10.1111/jcpe.12931

RANDOMIZED CLINICAL TRIAL

Human intrabony defect regeneration with micrografts

containing dental pulp stem cells: A randomized controlled
clinical trial

Francesco Ferrarotti | Federica Romano | Mara Noemi Gamba | Andrea

Quirico | Marta Giraudi | Martina Audagna | Mario Aimetti

Department of Surgical Sciences, C.I.R.

Dental School, University of Turin, Turin,
Italy
Correspondence
Mario Aimetti, C.I.R. Dental School, Via
Nizza 230 10126 Turin (Italy).
Email: mario.aimetti@unito.it
Abstract
Aim: The goal of this study was to evaluate if dental pulp stem cells (DPSCs) delivered
into intrabony defects in a collagen scaffold would enhance the clinical and radio-
graphic parameters of periodontal regeneration.
Materials and Methods: In this randomized controlled trial, 29 chronic periodontitis

Funding information
The study was self-­funded by the authors
and their institution, but Human Brain Wave
srl (Turin, Italy) provided free material to be
used in this study.
patients presenting one deep intrabony defect and requiring extraction of one vital
tooth were consecutively enrolled. Defects were randomly assigned to test or con-
trol treatments which both consisted of the use of minimally invasive surgical tech-
nique. The dental pulp of the extracted tooth was mechanically dissociated to obtain
micrografts rich in autologous DPSCs. Test sites (n = 15) were filled with micrografts
seeded onto collagen sponge, whereas control sites (n = 14) with collagen sponge
alone. Clinical and radiographic parameters were recorded at baseline, 6 and
12 months postoperatively.
Results: Test sites exhibited significantly more probing depth (PD) reduction (4.9 mm
versus 3.4 mm), clinical attachment level (CAL) gain (4.5 versus 2.9 mm) and bone
defect fill (3.9 versus 1.6 mm) than controls. Moreover, residual PD < 5 mm (93% ver-
sus 50%) and CAL gain ≥4 mm (73% versus 29%) were significantly more frequent in
the test group.
Conclusions: Application of DPSCs significantly improved clinical parameters of peri-
odontal regeneration 1 year after treatment.

KEYWORDS
dental pulp, periodontal pocket, periodontal regeneration, randomized controlled trial, stem
cells, tissue engineering

1 |  I NTRO D U C TI O N pivotal role in regeneration of tooth-­supporting tissues (Wikesjo

The goal of periodontal therapy is to arrest disease progression
and ultimately to regenerate lost periodontal tissues (Karring,
Nyman, Gottlow, & Laurell, 1993). Several studies over the past
30 years had demonstrated that blood clot stability plays a
& Nilveus, 1990; Wikesjo et al., 2003), avoiding apical migration
of epithelial cells during the first healing phase. In this respect,
new surgical techniques designed to optimize flap and clot sta-
bility (Cortellini & Tonetti, 2007, 2009; Harrel, Nunn, & Belling,
1999; Trombelli, Farina, Franceschetti, & Calura, 2009) and new

J Clin Periodontol. 2018;45:841–850. wileyonlinelibrary.com/journal/jcpe   © 2018 John Wiley & Sons A/S. |  841
Published by John Wiley & Sons Ltd
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842       FERRAROTTI et al.
biological agents promoting periodontal ligament and root ce-
mentum formation (Hammarström, 1997; Trombelli & Farina,
2008) have been developed. However, the ability to predictably
regenerate the periodontal damaged tissues still remains a major
unmet objective of regenerative therapies (Aichelmann-­Reidy &
Reynolds, 2008).
Tissue engineering (Palmer & Cortellini, Group B of the
European Workshop on Periodontology, 2008) has recently been
shown a promising approach for periodontal regeneration, and
strategies using adult mesenchymal stem cells (MSCs) are prom-
ising (Hynes, Menicanin, Gronthos, & Bartold, 2012; Peng, Ye, &
Zhou, 2009). Adult stem cells are undifferentiated cells found in
specialized tissues and organs of adults that have the capacity of
self-­renewal and the potential to differentiate in a multiple, but
limited, number of cells lineages (Lin, Gronthos, & Bartold, 2008).
Teeth have been demonstrated to represent an important MSCs
niche due to their embryogenic origin and postnatal development
(Huang, Gronthos, & Shi, 2009). MSCs have been isolated from
exfoliated deciduous teeth, apical papilla of immature permanent
teeth, dental follicle, periodontal ligament and dental pulp. Human
dental pulp stem cells (DPSCs) are especially attractive because
they are easy accessible, share the same origin and similar anti-
genic pattern of periodontal stem cells and can differentiate in the
same lineages (Gronthos et al., 2002; Huang et al., 2009; Tziafas
& Kodonas, 2010). They have a long lifespan, interact with bio-
materials and can be safely cryopreserved (Graziano et al., 2008a;
Papaccio et al., 2006).
Experimental evidence from in vivo studies and animal models
suggests that DPSCs have the capacity of producing lamellar bone
with appropriate vascularization and the potential to differentiate in
periodontal tissues (Graziano et al., 2008b; Khorsand et al., 2013).
Despite these encouraging results, the need for culture expand-
ing procedures to obtain a sufficient number of cells for delivery to
the damaged site increases the regulatory difficulties for translating
stem cells therapies to a clinical setting. Recently, autologous micro-
grafts rich in progenitor cells were obtained by mechanical disaggre-
gation of periosteum and dental pulp without the need for culture
expanding procedures. Micrografts displayed in vitro high cell via-
bility and optimal regenerative potential (Monti et al., 2017; Trovato
et al., 2015).
Some initial reports in humans demonstrated the clinical
and radiographic efficacy of dental pulp micrografts in postex-
traction alveolar defects (d’Aquino et al., 2009; Monti et al.,
2017) or in periodontal intrabony defects (Aimetti, Ferrarotti,
Cricenti, Mariani, & Romano, 2014; Aimetti, Ferrarotti, Gamba,
Giraudi, & Romano, 2018). These preliminary results suggest that
DPSCs may represent a promising tool for bone and periodontal
regeneration. Due to the lack of randomized controlled studies,
the aim of the study was to assess if dental pulp micrografts de-
livered into deep intrabony defects in a collagen scaffold would
enhance the clinical and radiographic parameters of periodontal
regeneration.
Clinical relevance
Scientific rationale for the study: Research is seeking to iden-
tify ways to improve regenerative therapy predictability.
This randomized and controlled study reports on the ap-
plication of dental pulp stem cells (DPSCs) in periodontal
regeneration in humans.
Principal findings: The application in intrabony defects of
DPSCs seeded onto a collagen scaffold enhances clinical
and radiographic parameters of periodontal regeneration
at 1-­year follow-­up.
Practical implications: DPSCs may represent a promising re-
generative procedure to treat intrabony defects in perio-
dontitis patients. The need for a vital and intact tooth
requiring extraction limits their clinical applicability.
2 | M ATE R I A L A N D M E TH O DS
2.1 | Study population and Experimental design
This article is reported according to the CONSORT statement. This
study was designed as a parallel, double-­
blind, prospective ran-
domized trial to evaluate the clinical and radiographic outcomes
12 months following two different surgical treatments of deep in-
trabony defects: minimally invasive surgical technique (MIST) plus
dental pulp micrografts in a collagen sponge biocomplex (test) ver-
sus MIST plus collagen sponge alone (control). The study was ap-
proved by the Institutional Ethical Committee (protocol n. 56/2016)
and conducted in accordance with the principles of Declaration of
Helsinki as revised in 2008. The trial was registered at clinicaltri-
als.gov as NCT03386877. Patients provided written consent for
participation.
Consecutive periodontitis patients who had completed nonsur-
gical periodontal treatment at the Section of Periodontology, C.I.R.
Dental School, University of Turin were screened for inclusion. The
following eligibility criteria were used: (a) diagnosis of advanced
chronic periodontitis (Armitage, 1999); (b) full-­mouth plaque score
(FMPS) and full-­mouth bleeding score (FMBS) < 15% at the time of
enrolment; (c) aetiological periodontal therapy completed at least
3 months prior to screening; (d) presence of one natural tooth hav-
ing a vertical defect with residual probing depth (PD) ≥ 6 mm and a
radiographic intrabony component ≥3 mm; (e) presence of one vital
and intact tooth requiring extraction due to impaction or malposi-
tion, as autologous source of DPSCs.
Exclusion criteria included: (a) smoking habits; (b) contraindi-
cations for periodontal surgery; (c) systemic diseases affecting
periodontal healing; (d) pregnancy and lactation; (e) history of peri-
odontal surgery or prosthetic restorations at the experimental teeth;
(f) clinical evidence of furcation defects (Hamp, Nyman, & Lindhe,
1975).
FERRAROTTI et al.
2.2 | Sample size and randomization
The difference in radiographic bone fill between test and control pro-
cedures was set as the primary outcome of the study. A difference
of 1.5 mm was assumed to be clinically significant and the amount of
variation was 1.1 mm (Cortellini & Tonetti, 2011). Therefore at 0.05
two-­sided alpha error and 80% power, the calculated sample size
was 24 patients (12 per group) that increased to 15 for compensation
of possible dropouts. Post hoc power calculation revealed that the
study had 90% power to detect differences in the primary outcome
measure.
After baseline visit, patients were randomized to the test or
control treatment using computer-­generated random sequence al-
location. To conceal assignment, forms with the treatment modality
were put into opaque and sealed envelopes with the patient number
on the outside. The envelopes were placed into the custody of the
study coordinator. The surgeon, the examiner who performed the
measurements and participants were blinded to treatment assign-
ment. Code breaking was performed after statistical analysis.
2.3 | Data collection
2.3.1 | Clinical measurements
The following clinical parameters were assessed on the experimental
teeth at baseline, 6 and 12 months after surgery using a periodontal
probe (PCP 15/11.5; Hu-­Friedy, Chicago, IL, USA): (a) presence/ab-
sence of plaque (PI 0/1); (b) presence/absence of BoP (0/1); (c) PD;
(d) gingival recession (REC); and (e) CAL, FMPS and FMBS were also
calculated as the percentage of gingival units (six sites per tooth)
around all teeth that revealed PI or BoP. During the surgical session
defect morphology was characterized in terms of the distance in mm
(INTRA) from the most coronal extension of the bone crest (BC) to
the bottom of the defect (BD) and the extent in mm of one, two and
three wall subcomponents of the defect.
One blinded and calibrated examiner (M.N.G.) carried out all
clinical measurements. For the calibration exercise, ten chronic peri-
odontitis patients not enrolled in the study were evaluated on two
separate occasions, 24 hr apart. Calibration was accepted if mea-
surements of PD and CAL at baseline and at 24 hr were similar to
the millimetre at ≥90%. The agreement was between 92% and 95%.
2.3.2 | Radiographic measurements
Periapical standardized radiographs were taken by a clinician masked
to the clinical measurements (M.G.) using the paralleling technique
and individually customized bite-­blocks (RINN XCP Film Holding
Instruments; Dentsply, York, USA) at baseline, 6 and 12 months after
surgery. The anatomical landmarks were identified as previously de-
scribed (Schei, Waerhaug, Lodval, & Arno, 1959). The radiographic
angle (RA) and the linear distance from BC and BD (intrabony de-
fect depth, IBD) were analysed using an image-­analysis software
(Image-­
J; NH). The difference between IBD values recorded at
|
      843
baseline and at 1-­year examination was identified as the amount of
radiographic bone fill.
2.4 | Surgical procedures
Two clinicians (M.A. and F.F.) were involved in the surgical sessions.
To ensure allocation concealment, one operator (M.A.) performed all
the surgical procedures, while the other one (F.F.) carried out tooth
extraction and filled the intrabony defects with the collagen sponge
provided by the study coordinator (A.Q.). Defects were accessed
with the MIST (Cortellini & Tonetti, 2007) using an operative micro-
scope (Zeiss S7; Feldbach, Switzerland). The elevation of the flap was
kept at minimum to allow the exposure of the defect and the care-
ful debridement of the root surface using manual (mini five Gracey
curettes; Hu-­Friedy, Chicago, IL, USA) and ultrasonic (Cavitron®
SelectTM; Dentsply) devices.
Following intrasurgical data registration, the tooth scheduled
for extraction was removed, washed in 0.2% chlorhexidine (CHX)
for 60 s and given to the study coordinator, charged for DPSCs iso-
lation. After opening the sealed envelope, he sectioned the tooth
from the test group along the CEJ to expose the pulp chamber and
collected the pulp tissue with a sterile Gracey curette. Then, the pulp
was mechanically dissociated using the Rigenera Machine System
(Rigenera®; HBW, Turin, Italy), a biological tissue disaggregator
working at a rotating speed of 80 rpm, in 1.0 ml sterile physiologic
solution (Aimetti et al., 2014; d’Aquino et al., 2009). After dissocia-
tion, the cellular suspension was passed through a disposable grid
(Rigeneracons) with 100 hexagonal holes filtering cells and compo-
nent of extracellular matrix with a cut-­off of 50 μm in an average
time of 90 s. The obtained micrografts enriched in DPSCs were en-
dorsed onto a collagen sponge scaffold (Condress®, Istituto Gentili,
Milano, Italy) to form a biocomplex. In the control group the collagen
sponge was only hydrated using physiologic sterile solution without
any addition of pulp cells.
The flaps were repositioned and tension-­free primary flap clo-
sure was obtained using horizontal internal mattress and interrupted
sutures.
2.5 | Postsurgical care
Patients were prescribed antibiotics (875 mg amoxicillin + 125 mg
clavulanic acid, 1 g b.i.d for 6 days) and analgesics (ibuprofen 600 mg,
if needed). They were advised to avoid toothbrushing and flossing in
the treated area for the first 2 weeks and to rinse with 0.12% CHX
solution three times a day for 4 weeks. After 2 weeks sutures were
removed and patients were instructed to use a soft toothbrush in the
surgical area. After 4 weeks, they discontinued CHX mouthrinse and
resumed conventional hygiene practices with medium toothbrush
and interdental devices.
Recall appointments were scheduled weekly during the first
month and every 3 months for the remainder of the observational
period. They consisted of reinforcement of oral hygiene mea-
sures, polishing, full-­mouth scaling and root planing and occlusal
 |
844       FERRAROTTI et al.
adjustment when needed. Subgingival instrumentation was not per-
formed in the treated area over 12 months.
2.6 | Statistical analysis
The primary outcome measurement was the radiographic bone fill.
Secondary outcome measurements included changes in clinical pa-
rameters. Only one defect per subject was included in the study.
Mean values and standard deviations (SD) were calculated for every
variable and for every assessment time point.
To test whether the data were normally distributed the
Kolmogorov–Smirnov test was done. The homogeneity of groups
F I G U R E   1   Consort diagram showing the study design
at baseline was tested using the unpaired t-­test (PD, CAL, IBD,
INTRA) and the Mann–Whitney U test (FMPS, FMBS, REC). The
repeated-­measures ANOVA was used to assess the effect of time
and treatment on continuous variables with a Gaussian distribution.
The Friedman test was applied to variables without a normal distri-
bution. Intragroup multiple comparisons were conducted with the
post hoc tests (Newman–Keuls test or Dunn test). The intergroup
differences were statistically explored using the unpaired Student
t-­test or the Mann–Whitney U test. The Bonferroni correction was
applied for multiple comparisons. The level of significance was set
at 5%. Statistical analyses were conducted using commercially avail-
able software (SAS version 9.2).
FERRAROTTI et al. |
      845

3 | R E S U LT S Changes in clinical parameters were significantly different be-

tween the two procedures (p ≤ 0.03). The test group showed greater
The flow chart of the experimental design is presented in Figure 1. CAL gain and PD reduction at both re-­evaluation assessments.
Twenty-­nine patients (13 males and 14 females), 39–69 year old The frequency distributions of residual PDs and CAL changes at
(mean age 50.7 ± 8.5 years), meeting the inclusion criteria were 12 months are summarized in Table 3. At 12-­month follow-­up visit,
treated. Fifteen bony defects (six maxillary and nine mandibular) re- complete pocket closure (PD ≤ 3 mm) was observed at a frequency
ceived MIST and dental pulp micrografts/collagen biocomplex, while of 66.7% in test sites and 14.3% at control sites. A CAL gain ≥4 mm
14 (eight maxillary and six mandibular) MIST and collagen sponge was measured in 73.3% and 28.6% of test and control intrabony de-
without cells. No subject discontinued participation in the study and fects, respectively.
no data points were missing for analysis. The first surgical procedure Radiographic data are summarized in Table 4. Statistically signifi-
was carried out in January 2016. All 12-­month follow-­up visits were cant differences between test and control group in IBD values were
completed in April 2017. observed at both 6-­(p < 0.001) and 12-­month follow-­up (p < 0.001).
Patient characteristics at baseline were not significantly differ- At 12 months, the mean radiographic bone fill was 3.9 ± 1.5 mm and
ent (p > 0.05) between groups (Table 1). The distributions of intra- 1.6 ± 1.1 mm in the test and control group, respectively. Two treated
bony defects according to teeth were: 20% incisive, 13.4% canine, cases are illustrated in Figures 2 and 3.
26.7% premolar and 40% molar for the test group and 28.6% inci-
sive, 21.4% canine, 14.3% premolar and 35.7% molar, for the control
group. As reported in Table 2, no statistically significant difference 4 | D I S CU S S I O N
was detected for any of the baseline defect characteristics between
test and control sites. In spite of the number of current available regenerative proce-
Mean ± SD of all clinical and radiographic parameters over the dures and biomaterials for the management of periodontal intra-
12-­month period are presented in Table 2. Primary closure was ob- bony defects, clinicians continue to seek more predictable and less
tained and maintained in all the sites. Soft tissues achieved complete technique-­sensitive regenerative therapies. Recent evidence from
healing within 3–4 weeks. No infectious episodes related to both several in vitro studies and animal models as well as from small-­scale
procedures were reported. FMPS and FMBS remained below 20% clinical pilot studies indicate that DPSCs may be a powerful tool for
through the study period in both groups, indicating a good standard bone and periodontal regeneration (d’Aquino et al., 2009; Khorsand
of plaque control. et al., 2013; Monti et al., 2017). To the best of our knowledge, this is
Both surgical techniques resulted in statistically significant the first randomized controlled study on the use of collagen sponge
overall changes in PD and CAL between baseline and 12-­month ex- scaffold complexed with autologous dental pulp micrografts in the
amination (p < 0.001). As shown in Table 2, the greatest PD reduc- regenerative treatment of deep intrabony defects with a prevalent
tion and CAL gain occurred during the first 6 months postsurgery noncontaining configuration.
(p < 0.001). The experimental therapy resulted in significantly more PD re-
In the DPSCs-­treated group mean PD reduction and mean CAL duction, CAL gain and radiographic bone gain than the open flap
gain amounted to 4.9 ± 1.4 mm and to 4.5 ± 1.9 mm during the 12-­ instrumentation, demonstrating the bioactivity of the micrografts
month period. A not statistically significant increase in REC values of
0.4 ± 1.1 mm was observed at the end of the experimental period.
TA B L E   2   Baseline characteristics of intrabony defect sites
In the control group mean PD reduction was 3.4 ± 1.7 mm and
mean CAL gain was 2.9 ± 2.2 mm at 12-­month follow-­up when com- Test group Control group
pared to baseline values. At this time point, a not statistically signifi- Variable (n = 15) (n = 14) p Value
cant REC increase of 0.5 ± 0.9 mm had taken place. PD (mm; mean ± SD) 8.3 ± 1.2 7.9 ± 1.3 0.380*
CAL (mm; 10.0 ± 1.6 9.4 ± 1.5 0.276 *
mean ± SD)
TA B L E   1   Characteristics of study subjects at baseline
INTRA (mm) 6.9 ± 1.5 6.3 ± 1.0 0.254*
Test group Control 1-­wall (%) 44.2 ± 24.8 37.7 ± 19.4 0.440**
Variable (n = 15) group (n = 14) p Value
2-­wall (%) 27.0 ± 13.5 36.0 ± 18.1 0.141**
Age (years; 51.9 ± 8.4 49.4 ± 9.3 0.404* 3-­wall (%) 28.8 ± 20.3 26.3 ± 16.4 0.726**
mean ± SD)
Radiographic angle 27.0 ± 4.8 25.9 ± 6.2 0.702*
Females/males (n) 7/8 8/6 0.853** (°; mean ± SD)
FMPS (%; mean ± SD) 10.9 ± 3.9 10.1 ± 2.1 0.470*** IBD (mm; mean ± SD) 6.4 ± 1.4 5.6 ± 1.0 0.115 *
FMBS (%; mean ± SD) 8.9 ± 3.7 9.6 ± 3.9 0.647***
Note. CAL: clinical attachment level; IBD: radiographic intrabony defect
Note. FMPS: full-­mouth plaque score; FMBS: full-­mouth bleeding score; depth; INTRA: intrasurgical intrabony defect depth; PD: probing depth;
SD: standard deviation. SD: standard deviation.
*Unpaired t-test. **Chi-­square test. ***Mann–Whitney U test. *Unpaired t-­test. **Mann–Whitney U test.
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846       FERRAROTTI et al.

TA B L E   3   Changes in clinical and radiographic parameters (mean ± SD) over the 12-­month experimental period

Variable Group Baseline 6 months Δ0–6 months 12 months Δ0–12 months

*
FMPS (%) Test 10.9 ± 3.9 10.0 ± 3.5 0.9 ± 6.1 9.7 ± 4.6 1.2 ± 6.7
*
Control 10.1 ± 2.1 11.6 ± 3.7 −1.5 ± 3.5 12.3 ± 5.1 −2.2 ± 5.7
 Difference between NSa NSb NSb
groups
FMBS (%) Test 8.9 ± 3.7* 7.5 ± 4.7 1.4 ± 4.1 7.7 ± 2.9 1.2 ± 4.5
*
Control 9.6 ± 3.9 8.1 ± 3.6 1.5 ± 3.6 7.4 ± 3.5 2.1 ± 5.4
 Difference between NSa NSb NSb
groups
PD (mm) Test 8.3 ± 1.2** 3.5 ± 0.8 4.8 ± 0.9*** 3.4 ± 0.9 4.9 ± 1.4***
Control 7.9 ± 1.3** 4.6 ± 1.0 3.3 ± 1.6*** 4.5 ± 1.0 3.4 ± 1.7***
a b b
 Difference between NS p = 0.002 p = 0.001
groups
CAL (mm) Test 10.0 ± 1.6** 5.4 ± 1.2 4.6 ± 1.4*** 5.5 ± 1.1 4.5 ± 1.9***
** ***
Control 9.4 ± 1.5 6.6 ± 1.3 2.8 ± 1.7 6.5 ± 1.2 2.9 ± 2.2***
 Difference between NSa p = 0.03b p = 0.01b
groups
REC (mm) Test 1.7 ± 1.2* 1.9 ± 1.2 −0.2 ± 0.6 2.1 ± 1.3 −0.4 ± 1.1
Control 1.5 ± 0.8* 2.0 ± 1.1 −0.5 ± 0.9 2.0 ± 1.2 −0.5 ± 0.9
a b b
 Difference between NS NS NS
groups
IBD (mm) Test 6.4 ± 1.4** 2.7 ± 0.8 3.7 ± 1.1*** 2.5 ± 0.7 3.9 ± 1.2***
** ***
Control 5.6 ± 1.0 4.1 ± 0.9 1.5 ± 1.2 4.0 ± 0.8 1.6 ± 1.1***
 Difference between NSa p < 0.001b p < 0.001b
groups

Note. CAL: clinical attachment level; FMBS: full-­mouth bleeding score; FMPS: full-­mouth plaque score; IBD: radiographic intrabony defect depth; NS:
difference between groups is not statistically significant (p > 0.05); PD: probing depth; REC: gingival recession.
a
Mann–Whitney U test or unpaired t-­test. bBonferroni-­corrected Mann–Whitney U test or Bonferroni-­corrected t-test.
*
p > 0.05, p values represent changes among the three time points (ANOVA or Friedman’s test). **p < 0.001, p values represent changes among the three
time points (ANOVA or Friedman’s test). ***p ≤ 0.001, p values represent longitudinal changes from baseline (Tukey test or Dunn test).

TA B L E   4   Intergroup comparison of frequency distribution of lack of periodontal stability (Lang & Tonetti, 2003; Matuliene et al.,
residual PD and CAL gain measured 1 year after treatment 2008, 2010).
Variable Category Test (%) Control (%) The achieved CAL gain in the test group (4.5 ± 1.9 mm) was
better than those reported in a systematic review by Graziani et al.
Residual PD (mm) 0–1 0 (0) 0 (0)
(2012) concerning the papilla preservation flap methods (2.48 mm,
2–3 10 (66.7) 2 (14.3)
CI: 1.44–3.52). The present findings are also more favourable with
4–5 5 (33.3) 10 (71.4)
respect to those reported in controlled clinical trials evaluating the
≥6 0 (0) 2 (14.3)
outcomes of regenerative surgery in intrabony defects using enamel
CAL gain (mm) 0–1 0 (0) 4 (28.4) matrix derivative and minimally invasive surgery (Cortellini & Tonetti,
2–3 4 (26.7) 6 (42.9) 2009; Jepsen et al., 2008; Ribeiro, Casarin, Jùnior, Sallum, & Casati,
4–5 6 (40) 3 (21.4) 2011). At 12 months they reported CAL changes ranging from 1.8
≥6 5 (33.3) 1 (7.1) to 4.9 mm and PD reduction from 2.6 to 5.2 mm. Notably, all DPCS-­

Note. CAL: clinical attachment level; PD: probing depth.
and enhancement of the intrinsic healing potential in the human in-
trabony defect environment. The 93% of the test sites compared to
50% of control sites exhibited residual PD ≤ 4 mm at 12-­month fol-
low-­up. Several studies reported the association between presence
of residual pockets after treatment, especially with PD ≥ 5 mm and
treated sites were filled by radiographic bone-­like tissue with a mean
bone fill of 3.9 ± 1.2 mm.
Consistent with the results of previous studies, the magni-
tude of changes in CAL (2.9 ± 2.2 mm) and PD (3.4 ± 1.7 mm) were
more modest in the control group and inferior to the regenerative
technique evaluated in parallel (Graziani et al., 2012; Needleman,
Worthington, Griedys-­
Leeper, & Tucker, 2006; Tu, Needleman,
Chambrone, Lu, & Faggion, 2012).
FERRAROTTI et al. |
      847

(a) (b) (c) (d)

(e) (f) (g) (h)

F I G U R E   2   Test site treated with minimally invasive surgical technique and autologous dental pulp micrografts/collagen sponge
biocomplex (periodontal surgeon M.A.). Preoperative radiographic view of an intrabony defect on the distal aspect of the maxillary first
molar (a), elevation of the flap and probing of the intrabony defect (b), intraoperative view of the defect before elevating the interdental
papilla (c), mechanical dissociation of the dental pulp (d), placement of a collagen sponge with dental pulp micrografts (e), suture (f), clinical (g)
and radiographic (h) aspects at 12 months after treatment (h)

(a) (b) (c)

(d) (e) (f)

F I G U R E   3   Control site treated with minimally invasive surgical technique and collagen sponge alone. Preoperative radiographic (a) and
clinical views (b) of an intrabony defect on the distal aspect of the mandibular first premolar, elevation of the flap and defect debridement (c),
suture (d), radiographic (e) and clinical (f) aspects at 12 months after treatment
 |
848       FERRAROTTI et al.
In recent years several therapeutic protocols for autologous
human stem cells transplantation have been conducted with differ-
ent results (Hynes et al., 2012). The surgical access to these poten-
tial collection sites often represents a limiting point and the ratio
between tissue collected and stem cells isolated is often disadvan-
tageous. Dental pulp is a niche housing neural crest-­derived stem
cells. It is easily accessible and there is limited morbidity after col-
lection (Mitsiadis, Barrandon, Rochat, Barrandon, & De Bari, 2007).
Recently, a novel method of cells extraction based on mechanical
disaggregation of connective tissues has been introduced (d’Aquino
et al., 2009). It produces in a few minutes millions of micrografts
from a few millimetres sample of autologous dental pulp and filters
them with a cut-­off of 50 μm in order to promote the discharging
of old differentiated cells and the enrichment of young progenitor
cells (Monti et al., 2017; Trovato et al., 2015). Under this dimension
the percentage of cells expressing stem antigens grows dramatically,
avoiding a magnetic or flow cytometric sorting (Iohara et al., 2006).
Dental pulp stem cells are fully comparable to cells obtained
after enzymatic digestion but they are produced directly within
the surgery and immediately used without any manipulation (Monti
et al., 2017). They showed cell viability of 92% with 7AAD staining,
tended to express surface markers of mesenchymal cells such as
STRO-­1, CD44 and CD90 and were able to differentiate in adipo-
cytes, osteocyte and chondrocytes lines in vitro (Monti et al., 2017).
Flow cytometry analysis demonstrated that human dental pulp mi-
crografts are highly positive to Flk-­1 and CD34, markers of stromal
and vascular endothelial progenitors, and contain about 77% of pro-
genitors cells, a sufficient quantity to perform in vivo experiments
(d’Aquino et al., 2009). Pericytes are a heterogeneous population of
mesenchymal cells associated with the microvasculature, which vary
greatly in origin, morphology, surface markers and function in differ-
ent tissues. Some pericyte markers are also expressed on other cell
types, most notably endothelial and smooth muscle cells (Armulik,
Genové, & Betsholtz, 2011). Similar to other types of stem cells, peri-
cytes act as a repair system in response to injury by maintaining the
structural integrity of blood vessels (Wong et al., 2015). The CD34
and Flk-­1 cytotype seems to have a very interesting power to induce
healing and regeneration process and to be the much suitable cell
population for clinical approach (Graziano et al., 2008b).
Previous investigations in the canine model demonstrated that
DPSCs have the ability to regenerate periodontal tissues in surgically
created intrabony defects (Khorsand et al., 2013) and to form new
cementum in furcation perforations (Bakhtiar et al., 2016).
The clinical efficacy of this method for DPSCs collection has
been demonstrated in humans in sinus lift procedures and in bone
regeneration of postextraction alveolar defects (d’Aquino et al.,
2009; Brunelli et al., 2013; Graziano, d’Aquino, Brunelli, Fanali, &
Carinci, 2011). Preliminary data are available for periodontal regen-
eration of intrabony defects (Aimetti et al., 2014, 2018).
The same mechanical disaggregation protocol has been used in
the present investigation to obtain a cellular suspension endorsed
onto collagen scaffold. Collagen sponge supports stem cells pro-
liferation and differentiation within the first weeks and stimulates
the formation of calcified tissues (Sumita et al., 2006). Mechanical
stability of the coagulum is a key factor for cell differentiation, and
it is pivotal when the committed tissue is a mineralized tissue like
the root cementum or the alveolar bone. Furthermore, collagen is
radiolucent, inactive in terms of periodontal regeneration and with a
rapid resorption rate.
An important aspect to take into account when interpreting the
results is that this technique, requiring a vital and intact tooth as
autologous donor site of DPSCs, has a limited applicability. There is
preliminary evidence that periodontally involved teeth in aggressive
periodontitis patients may contain putative stem cells with certain
MSC properties (Sun et al., 2014). This may widen the clinical appli-
cability of this procedure.
When extracting human teeth, another therapeutic option to
reach periodontal regeneration is to use autologous periodontal
ligament stem cells (PDLSCs). These cells exhibit multipotency, as
demonstrated by their ability of differentiating into osteoblasts, fi-
broblasts and cementoblasts and forming cementum and periodontal
ligament tissue (Seo et al., 2004). However, current PDLSCs-­based
periodontal cell therapies require ex vivo expansion with animal
serum in order to produce sufficient cell number before implantation
in human intrabony defects (Chen et al., 2016; Feng et al., 2010).
This raises some concerns on immunological risks and limits PDLSCs
applicability in periodontal clinical practice (Shahdadfar, Fronsdal,
Haug, Reinholt, & Brinchmann, 2005). In contrast, dental pulp pro-
vides sufficient tissue for selection of progenitor cells without the
need of prior culture expanding procedures, leading to easier appli-
cation in cell-­based periodontal therapy (Graziano et al., 2008b).
There are some limitations of this study. First, the sample size
was small and data were from one institution. Second, it is not possi-
ble to confirm that periodontal regeneration had indeed occurred to
the treated sites, because no histological analysis can be presented
for ethical reasons. However, radiographic bony changes have been
considered a valid parameter to clinically demonstrate the effective-
ness of regenerative procedures (Cortellini, Pini Prato, & Tonetti,
1993). Moreover, the selected intrabony defects had limited self-­
regenerative potential and were filled with a biologically inert and
radiolucent biomaterial.
5 | CO N C LU S I O N S
Despite these limitations, the study findings add significantly to the
existing literature on the clinical applicability of tissue engineering
to regenerative therapies. In individuals with one or more vital teeth
requiring extraction, the application of autologous dental pulp mi-
crografts into deep noncontaining intrabony defects coupled with
minimally invasive surgical procedures, to optimize blood stability
and to provide a stable space for regeneration, could enhance the
intrinsic regenerative potential of the periodontal defect. In this way,
using this protocol, the patient is, at the same time, the donor and
the acceptor of DPSCs, and this system permits to increase the pro-
genitor cell viability in the surgical site that needs to be regenerated.
FERRAROTTI et al.
This could limit the need for grafting materials and barrier mem-
branes. Independent clinical trials are needed to confirm the promis-
ing results of the present study.
C O N FL I C T O F I N T E R E S T
The authors report no conflict of interests related to this study.
ORCID
Mario Aimetti  http://orcid.org/0000-0003-0657-0787
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How to cite this article: Ferrarotti F, Romano F, Gamba MN,
et al. Human intrabony defect regeneration with micrografts
containing dental pulp stem cells: A randomized controlled
clinical trial. J Clin Periodontol. 2018;45:841–850. https://doi.
org/10.1111/jcpe.12931