Bone Formation Following Sinus Augmentation with an

Equine-Derived Bone Graft: A Retrospective Histologic and

Histomorphometric Study with 36-Month Follow-up
Danilo Alessio Di Stefano, DDS1/Giorgio Gastaldi, MD, DMD2/Raffaele Vinci, MD, MFS, DMD3/
Elisabetta Maria Polizzi, BS, DH4/Lorenzo Cinci, PhD5/Laura Pieri, PhD6/ Enrico Gherlone, MD, DMD7

Purpose: The aim of this study was to investigate bone formation over time following maxillary sinus augmentation
with an enzyme-deantigenic, bone collagenpreserving equine bone graft by retrospective assessment of
histomorphometric data. Materials and Methods: Records of patients with atrophic ridges who underwent maxillary
sinus augmentation with the enzyme-deantigenic equine bone graft and two-step implant placement between
3 and 12 months after the sinus-augmentation surgery were assessed retrospectively. The histomorphometric
data were clustered in three classes according to time of collection from the augmentation surgery and analyzed
to assess newly formed bone deposition and residual biomaterial degradation rates. Data concerning the
36-month clinical follow-up were also assessed. Results: Records of 77 patients and 115 biopsy specimens
were retrieved, and histomorphometric data were clustered (3 to 5 months, n = 33; 6 to 8 months, n = 57;
9 to 12 months, n = 25). Mean minimum atrophic ridge thickness was 4.9 0.5 mm (range, 4.0 to 7.1 mm).
The amount of newly formed bone and residual biomaterial did not significantly differ among the three clusters.
Qualitative analysis showed a denser trabecular structure in late (> 8 months) samples. At the 36-month clinical
follow-up, no differences were found among the implant success rates in the three groups, according to the
Albrektsson and Zarb criteria for success. The overall implant success rate was 98.3%. Conclusion: Based
upon this retrospective human study of 77 patients with 4 to 7 mm of residual bone, when enzyme-deantigenic
equine bone is used for sinus augmentation, new bone formation occurs at an early time (< 3 months) after the
grafting, and implant placement can be safely carried out as soon as 3 to 5 months after the augmentation
surgery. Int J Oral Maxillofac Implants 2016;31:406412. doi: 10.11607/jomi.4373

Keywords: biomaterial, bone remodeling, sinus augmentation, xenograft

S inus augmentation is a predictable surgical proce-

dure for the prosthetic rehabilitation of the atrophic
maxilla. As it involves placing graft material in a four-wall
1 Adjunct Professor, Dental School, Vita-Salute University and
IRCCS San Raffaele, Milan, Italy.
2 Associate Professor, Dental School, Vita-Salute University and
IRCCS San Raffaele, Milan, Italy.
3Adjunct Professor, Dental School, Vita-Salute University and
IRCCS San Raffaele, Milan, Italy.
4 Adjunct Professor, Dental School, Vita-Salute University and
IRCCS San Raffaele, Milan, Italy.
5Researcher, Department of Neuroscience, Psychology, Drug
Research and Child Health (Neurofarba), Pharmacology and
Toxicology Section, University of Florence, Florence, Italy.
6Department of Health Sciences, Interdepartmental Forensic
Medicine Section, University of Florence, Florence, Italy.
7Full Professor and Chairman, Dental School, Vita-Salute
University and IRCCS San Raffaele, Milan, Italy.
Correspondence to: Prof Danilo Alessio Di Stefano, Via Matteo
Civitali 40, 20148 Milan, Italy. Fax: +39 02 48705638.
Email: distefano@centrocivitali.it
2016 by Quintessence Publishing Co Inc.
confined cavity, it has often been used as a model clinical
setting for collecting histomorphometric data concern-
ing different biomaterials.1 Accordingly, a great body
of literature about the performance of different bone
substitutes grafted in sinus-augmentation surgeries has
been published.2 Autogenous bone is regarded as the
gold standard among graft materials, as it contains both
active bone cells (osteoblasts, osteocytes, stem cells) and
growth factors that may prompt bone regeneration.3
However, the additional surgery needed for collecting
the graft material increases both morbidity and the risk
of intra- and postsurgical complications.46 To overcome
these limitations, other allogeneic, synthetic, and natural
biomaterials have been proposed as bone substitutes.7
Xenografts, ie, materials obtained by processing the
bone of a nonhuman mammal species to eliminate an-
tigens, have been subjected to intensive investigation
because of the features of their architecture and min-
eral composition that they share with human bone.8,9
Among xenografts, a form of enzyme-deantigenic
equine bone has been used successfully for bone recon-
struction both in fields different from oral surgery10 and

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in a wide range of oral applications, including periapical
cyst-removal management, periodontal defect correc-
tion, horizontal and vertical atrophic ridge reconstruc-
tion,1116 and sinus augmentation.1720 The enzymatic
process used to make the equine bone nonantigenic
preserves type I collagen, the most abundant protein of
bone extracellular matrix,21 in its native conformation.
This makes enzyme-deantigenic equine bone substan-
tially different from other xenografts, such as anorganic
bovine bone and others2225 that are obtained by elimi-
nating all protein components26 and other antigens
through high-temperature bone processing. This differ-
ence seems to affect cellular behavior. Indeed, when os-
teoclasts were cultured on enzyme-deantigenic equine
bone, they adhered on this material in greater numbers
and exerted a more intense degrading activity27 than
they did under the same experimental conditions on
anorganic bovine bone,28 possibly because of the pres-
ence of preserved collagen in the equine xenograft.29
This behavior that enzyme-deantigenic equine
bone shows in vitro, together with other effects that
preserved bone collagen could exert on bone cells and
biologic mechanisms related to bone regeneration,3036
could allow for faster formation of new bone. Consis-
tent with this hypothesis, a randomized clinical trial
that analyzed biopsy specimens collected 6 months
after sinus-augmentation surgery found that enzyme-
deantigenic equine bone led to a significantly greater
amount of newly formed bone and a lower residual
biomaterial, compared with anorganic bovine bone.37
A possible consequence of the higher bone deposi-
tion rate observed in sinuses augmented with enzyme-
deantigenic equine bone could be the possibility of
placing implants earlier than the standard 6-month
healing period. The aim of this retrospective study was
therefore to investigate the bone-deposition rate and
resorption properties of this equine biomaterial over
time and to evaluate possible correlations between the
time of implant placement from grafting surgery and
implant survival and success.
MATERIALS AND METHODS
Data Collection
Clinical records were selected among those of patients
with atrophic ridges referred to the Department of
Dentistry, San Raffaele Scientific Institute, Milano, Italy,
between January 2009 and December 2010 seeking
implant-supported rehabilitation of maxillae. Patients
included in the present retrospective study (1) under-
went sinus augmentation with enzyme-deantigenic
equine bone (Osteoxenon, Bioteck), (2) had delayed
implant placement, and (3) had at least one biopsy
specimen collected at the time of implant placement.
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Di Stefano et al
Other inclusion criteria were: an age between 35
and 70 years; the presence of partial or total edentu-
lism; and the lack of any systemic diseases. Patients
were eligible for regenerative treatment if they did
not present any of the following: pregnancy; osteo-
porosis, neoplasia, or psychiatric disease; acute oral
infections; coagulation disorders; history of chemo-
therapy or radiotherapy in the head or neck region;
immunocompromised status; current bisphospho-
nate therapy; chronic alcohol or drug abuse; or smok-
ing more than 10 cigarettes per day.
Surgical Procedure
After clinical examination and radiographic assess-
ment through orthopantomography and cone beam
computed tomography (CBCT), surgery was performed
as follows. Antibiotic prophylaxis (amoxicillin/clavu-
lanic acid, Augmentin, Glaxo-SmithKline; 2 g 1 hour
before surgery and then every 12 hours for 8 to 10
days) was initiated, and the patients were subjected
to mouthrinses with chlorhexidine 0.2% (Corsodyl,
Glaxo-SmithKline), to be continued for 2 weeks after
surgery. Nimesulide 100 mg (Aulin, Roche) was also
administered 1 hour before surgery and then twice
a day for 7 days.
The surgical area was anesthetized using articaine
hydrochloride 40 mg/mL with epinephrine 1:100,000.
Two experienced surgeons (RV, DDS) performed sinus
augmentation according to the technique described
by Boyne and James.3840 Full-thickness flaps were
elevated, and an access window was created on
the lateral wall of the maxillary sinus, using a round
diamond bur under irrigation with sterile saline. The
sinus membrane was carefully elevated, and the
window wall gently pushed inside the cavity, allow-
ing access into it. The enzyme-deantigenic equine
bone particles were hydrated with sterile saline and
inserted into the cavity, applying gentle pressure to
stabilize them.
A collagen membrane (Biocollagen, Bioteck) was
placed over the window, and mucoperiosteal flaps
were sutured using nonresorbable 5.0 sutures. Sutures
were removed after 10 days.
At the second surgery, all patients received at
least one implant in the grafted maxillary site. After
antibiotic prophylaxis and anesthesia were initiated, a
full-thickness flap was raised, the bone was inspected,
and bone cores were obtained from the occlusal
aspect of the alveolar ridge using a trephine drill
under irrigation with sterile saline (Fig 1). All biopsy
specimens were approximately 3 mm in diameter and
10 mm in length and were marked on the occlusal
side for the purpose of orientation during histologic
processing. Titanium implants, 3.8 to 5.0 mm wide and
10 to 13 mm long, were placed at the biopsy sites.
The International Journal of Oral & Maxillofacial Implants 407
Di Stefano et al
Alveolar Ridge Height Analysis and Statistics
Preoperative CBCT scans were independently analyzed
by two physicians (GG, EMP), who measured the residual
vertical ridge height at the site of implant insertion. Each
operator compared the postoperative radiograph with
the preoperative CBCT scans to determine on the latter
the position of implant insertion, and measured the ridge
height twice with the aid of the software provided with
the CBCT device (WhiteFox Control, Acteon). Measure-
ments of both operators were averaged to calculate
a single ridge height estimate corresponding to each
biopsy specimen and histomorphometric dataset. Data
were clustered in three groups according to the time
of biopsy specimen collection after the augmentation
surgery (3 to 5, 6 to 8, and 9 to 12 months). For each
group, the average bone height was calculated. A one-
way analysis of variance (ANOVA) was performed to
test for significant differences in the preoperative ridge
height among the three clusters. The significance level
of the test was .05.
Histologic and Histomorphometric
Assessment
Each bone core was placed in a test tube containing
buffered 10% formalin. The tube was marked with a
unique alphanumeric code and sent to the histologists
(LC, LP). Bone cores were decalcified for 21 days in a
0.76 mol/L sodium formate and 1.6 mol/L formic acid
solution (Panreac Quimica). Samples were subsequently
dehydrated in ascending concentrations of ethanol and
embedded in paraffin.
The bone cores were cut into 5-m-thick sections,
mounted on slides, and stained with hematoxylin-eosin.
One histologist (LC) provided, for each sample, a qualita-
tive report aimed at providing a general sample descrip-
tion and identifying any sign of inflammatory or immune
reactions. Slides were observed under polarized light
to better identify lamellar (mature), mineralized bone.
Morphometric measurements were performed on
digital photomicrographs collected at 10 magnification.
Each whole sample image was analyzed by two opera-
tors independently and in triplicate using the Image J
1.33 analysis software (National Institutes of Health). For
each image, the total sample area (TSA), the total bone
area (TBA), the newly formed bone area (LBA), and the
residual bone substitute area (RBA) were measured. Aver-
age newly formed bone (NFB) and residual biomaterial
(RB) were then calculated and expressed as the percent-
age over the total sample area (%NFB = LBA 100/TSA;
%RB = RBA 100/TSA). NFB and RB are given as the
mean percentage of all sections.
Histomorphometric Analysis and Statistics
NFB and RB data were clustered in three intervals ac-
cording to the time of biopsy specimen collection after
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augmentation surgery (3 to 5, 6 to 8, and 9 to 12 months).
For each interval, average NFB and RB were calculated
and plotted versus time.
A one-way ANOVA was performed to test for sig-
nificant differences in the bone-formation rate and
residual biomaterial remodeling among different time
clusters. The significance level of the test was .05. A
dedicated software program (Origin 9.0, Microcal) was
used for all statistical analyses. All values are presented
as mean standard deviation (SD).
Follow-up
After definitive prosthetic rehabilitation, patients were
recalled for control visits every 6 months up to 36 months.
At each visit, they received professional hygiene and a
thorough oral examination. Radiographs were collected
at the 12, 24, and 36-month follow-up controls.
Implant Survival and Success Rate
Implant success was evaluated according to criteria
described by Buser et al41 and modified by Albrekts-
son and Zarb42 and including: (1) absence of persistent
pain, dysesthesia, or paresthesia in the implant area;
(2) absence of peri-implant infection with or without
suppuration; (3) absence of perceptible mobility of the
implant; (4) absence of peri-implant bone resorption
greater than 1.5 mm during the first year of loading
and 0.2 mm/year in the following years. Implants were
considered successful when all the aforementioned
conditions were met.
RESULTS
Records were analyzed for 77 nonconsecutive pa-
tients (34 men and 43 women) with a mean age of
62.04 6.12 years (range, 52 to 70 years). All patients
completed the healing period following the sinus-
augmentation procedure with no symptoms of maxillary
sinusitis or other complications.
Residual Alveolar Ridge Height Analysis
Mean minimum preoperative ridge height value was
4.9 0.5 mm (n = 77; range 4.0 to 7.1). Mean minimum
ridge height for patients belonging to the three time
clusters was: 5.1 0.4 mm (n = 33, 3 to 5 months);
4.9 0.6 mm (n = 57, 6 to 8 months); 4.8 0.5 mm (n = 25,
9 to 12 months). No significant difference (P = .33) was
observed among the three clusters, meaning they were
homogenous as far as the preoperative height of the
alveolar ridge at the augmentation site was concerned.
Histologic Analyses
Extensive bone structures were present in all samples
(Figs 1 to 3). Residual particles of xenograft material
 Di Stefano et al

NFB NFB
RB RB

NFB NFB
RB RB
a b c
Fig 1 Alveolar bone biopsy sample collected 3 to 5 months after the grafting surgery. (a) Hematoxylin-eosin staining of the speci-
men showing the presence of newly formed bone (NFB) and residual biomaterial (RB). (b) The Image J 1.33 analysis software outlines
the areas on the section corresponding to NFB (turquoise) or RB (green). (c) The software fills the areas with false colors and calcu-
lates the ratio of all the areas of the same color to the total sample area. All the pictures were taken at 4 magnification.

NFB NFB

NFB NFB NFB NFB

RB RB RB RB

a b c
Fig 2Alveolar bone biopsy sample collected 6 to 8 months after the grafting surgery. NFB = newly formed bone (turquoise);
RB = residual biomaterial (green).

RB RB
RB RB
NFB NFB

RB
RB
a b c
Fig 3 Alveolar bone biopsy sample collected 9 to 12 months after the grafting surgery. This sample shows a denser trabecular
structure compared with the ones collected earlier. NFB = newly formed bone (turquoise); RB = residual biomaterial (green).

were identified as hematoxylin-stained areas in which
the bone lacunae were devoid of osteocytes. Being of
animal origin, their microscopic appearance resembles
that of human bone, though osteocytes have been
eliminated by the antigen-elimination process. They
were in close contact with the living, patient-derived
bone tissue (eosin-stained and osteocytes-rich). In
most samples, the medullary spaces were filled with
well-vascularized connective tissue and displayed no
areas with cartilage-like tissue. Osteoblasts could be
observed lining both the bone substitute and the newly
formed bone. Lamellar bone could be observed in most
of the samples; a more dense trabecular structure was
observed only in biopsy specimens collected 8 months
or later after the augmentation surgery. In all samples,
no sign of inflammation was detected.
Histomorphometric Analysis
Photographs of the histomorphometric mea-
surements are shown in Figs 1 to 3. The amount
of NFB and RB for the three time clusters were
NFB = 38.42% 14.24%; RB = 9.19% 6.60% (n = 33, 3 to 5
months); NFB = 37.77% 14.70%; RB = 11.65% 8.43%
(n = 57, 6 to 8 months); NFB = 42.68% 17.38%;
RB = 10.01% 8.22% (n = 25, 9 to 12 months). The cor-
responding plots are displayed in Fig 4.
No significant differences in NFB (P = .60) and in RB
(P = .22) percentages were observed among the three
clusters.
Follow-up Controls
According to Albrektsson and Zarbs criteria,42 two
implants were classified as failed, as they showed a
peri-implant resorption greater than 0.2 mm (0.4 and
0.5 mm, respectively) in the past 12 months, leading to a
success rate of 98.3%. The two failed implants were placed
respectively at 6 and at 8 months after augmentation
surgery. No patients suffered from prosthetic problems
during the follow-up controls.

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Di Stefano et al
70
60
50
NFB (%)
40
30
20
10
0 0
35 68 912
a Time (mo)
Fig 4 Histomorphometric analysis results. The percentages of (a) newly formed bone (NFB) and (b) residual biomaterial (RB) found
in bone samples at the three time intervals were not statistically significantly different.
DISCUSSION
Studies of animals have shown that the rate of bone
formation in grafted sinuses depends on the biomaterial
being grafted. Such differences have been observed both
at early and late stages of regeneration.4345 However, clini-
cal studies show that differences in the bone-formation
rate may be less evident, or even nonsignificant, when
samples are collected at 6 months, the usual healing time
for two-stage implant placement after sinus augmenta-
tion.4649 Also, after healing times longer than 6 months,
no significant differences in newly formed bone quantities
were detected,5052 unless autogenous bone was grafted
alone,53,54 in which case a greater volumetric bone loss
has been reported over time.5557 Mixing autogenous
bone with biomaterials does not show, at 6 months
from surgery, that it provides a significant increase in
bone formation compared with biomaterials alone.58,59
The present study retrospectively analyzed the histo-
logic and clinical outcomes at different times after maxillary
sinus augmentation using a collagen-preserving enzyme-
deantigenic equine bone. From a quantitative point of
view, no significant differences could be detected between
samples collected at different times as far as both NFB
and RB were concerned. It is known that the individual
bone-regeneration rate may differ significantly among
patients because of metabolic, anatomical, or habit dif-
ferences.6062 In this study, at all time points considered,
including the earlier ones, the dispersion of measurements
(quantified by the SD of each mean) was approximately
37% to 40%, meaning that an actual difference in the early
bone deposition rate could have been hidden by such
variability, and may have required a greater number of
samples per time point to be detected. Nonetheless, the
absence of a significant difference suggests that new bone
quantity may already reach a plateau at 3 to 5 months
after surgery, implying that the deposition of most of the
newly formed bone occurred earlier.
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30
20
RB (%)
10
35 68 912
b Time (mo)
This hypothesis is consistent with results from a
clinical study on a different graft material, mimicking
the cell-binding domain of type I collagen responsible
for cell migration, differentiation, and proliferation.63
The work showed that the newly formed bone fraction
in specimens retrieved from augmented sinuses after 2
months was not significantly different from that mea-
sured at 4, 6, and 9 months. Similarly, a study in which
a porcine-derived, collagen-preserving biomaterial was
grafted showed no significant differences in the rate of
bone deposition between biopsy specimens collected
at 4 and 6 months.64
Early deposition is in line with the significant differ-
ence observed in the amount of newly formed bone
that enzyme-deantigenic equine bone provided in sinus
augmentation, compared with anorganic bovine bone,37
which contains no collagen. Early bone deposition is
also consistent with previous studies showing that the
expression of markers typical of bone regeneration was
not significantly different when non-heat-treated equine
bone grafts18,20 or autogenous bone were used for sinus
augmentation, suggesting that heat treatment may alter
the behavior of the graft either by denaturing collagen
or by altering the bone mineral component. Consistent
with this hypothesis, when a heat-treated equine bone
graft was used for sinus augmentation,65 the amount of
newly formed bone was again similar to the one observed
when grafting anorganic bovine bone.37
From a qualitative point of view, no inflammatory reac-
tions could be observed, suggesting a neutral interaction
of the grafted particles with the recipient bone tissue at
all time points. The qualitative analysis of later samples
(9 to 12 months) showed a denser trabecular structure
than earlier ones, and a prevalence of woven versus
nonwoven bone, even with a high degree of variability
among samples. Overall, the observations of the present
study are compatible with a scenario in which new bone
deposition and biomaterial resorption occurred early

and therefore could not be directly observed, whereas
remodeling and bone organization in trabeculae was
still ongoing at later times.
Concerning clinical results, no significant differences
were observed in the survival rates between implants
placed at earlier and later times. These results are con-
sistent with recent studies showing that early implant
success and survival rates do not differ significantly
even between a one- or two-stage sinus-elevation pro-
cedure with anorganic bovine bone in patients with a
1- to 3-mm-thick residual ridge.66 Therefore, late implant
placement may not bring any clinical advantage in
terms of implant success or survival, at least in a short
or medium observation period. The success of implant
restoration could also be affected by the presence of
residual bone-graft material that modified the bones
load-bearing capabilities67 or interfered with the first
steps of osseointegration.68 This may be important,
for example, when bone grafting is carried out during
one-stage implant placement and, even more, when
immediate loading is planned. Under such conditions,
it might be desirable for the graft materials to undergo
remodeling for the faster formation of a biomechanically
strong tissue at the bone-implant interface, favoring
osseointegration. Further studies should therefore be
undertaken to clarify whether, after grafting enzyme-
deantigenic equine bone, long-term results (> 5 years)
concerning implant success and survival rates still remain
the same even if the time for implant placement after
sinus augmentation is shortened by 3 months.
CONCLUSIONS
Within the limits of a retrospective analysis and the
short follow-up observation period, the results of the
present study show that when enzyme-deantigenic
equine bone is used for sinus augmentation, new bone
formation occurs at an early time (< 3 months) after the
grafting surgery. Accordingly, implant placement in
two-stage procedures could be successfully performed
at 3 to 5 months.
ACKNOWLEDGMENTS
The authors reported no conflicts of interest related to this study.
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