biomimetics

Article
Influence of Trabecular Bone Presence on Osseodensification
Instrumentation: An In Vivo Study in Sheep
Zachary Stauber 1 , Shangtao Wu 1 , Justin E. Herbert 2 , Amanda Willers 3 , Edmara T. P. Bergamo 4 ,
Vasudev Vivekanand Nayak 2 , Nicholas A. Mirsky 1 , Arthur Castellano 5,6 , Sinan K. Jabori 7 , Marcelo V. Parra 8,9 ,
Estevam A. Bonfante 10 , Lukasz Witek 4,11,12, * and Paulo G. Coelho 2,7

1 University of Miami Miller School of Medicine, Miami, FL 016960, USA

2 Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine,
Miami, FL 33136, USA
3 Operative Dentistry Division, Department of Restorative Dentistry, Piracicaba Dental School, Piracicaba, State
University of Campinas, Piracicaba 13414-903, Brazil
4 Biomaterials Division, NYU Dentistry, New York, NY 10010, USA
5 Mackenzie Evangelical School of Medicine Paraná, Curitiba 80730-000, Brazil
6 Federal University of Parana, Curitiba 80060-000, Brazil
7 Division of Plastic Surgery, DeWitt Daughtry Family Department Surgery, University of Miami Miller School
of Medicine, Miami, FL 33136, USA
8 Center of Excellence in Morphological and Surgical Studies (CEMyQ), Faculty of Medicine, Universidad de la
Frontera, Temuco 01145, Chile
9 Department of Comprehensive Adult Dentistry, Faculty of Dentistry, Universidad de la Frontera,
Temuco 01145, Chile
10 Department of Prosthodontics and Periodontology, Bauru School of Dentistry, University of Sao Paulo,
Bauru 17012-901, Brazil
11 Hansjörg Wyss Department of Plastic Surgery, NYU Grossman School of Medicine,
New York, NY 10016, USA
12 Department of Biomedical Engineering, NYU Tandon School of Engineering, 6 MetroTech,
Brooklyn, NY 11201, USA
* Correspondence: lukasz.witek@nyu.edu

Citation: Stauber, Z.; Wu, S.; Herbert, Abstract: Osseodensification enhances the stability of endosteal implants. However, pre-clinical
J.E.; Willers, A.; Bergamo, E.T.P.; studies utilizing osseodensification instrumentation do not account for the limited presence of trabec-
Nayak, V.V.; Mirsky, N.A.; Castellano,
ulae seen clinically. This study aimed to evaluate the effect of osseodensification instrumentation on
A.; Jabori, S.K.; Parra, M.V.; et al.
osteotomy healing in scenarios with and without the presence of trabecular bone. A ~10 cm incision
Influence of Trabecular Bone Presence
was made over the hip of twelve sheep. Trabecular bone was surgically removed from twelve sites
on Osseodensification Instrumentation:
An In Vivo Study in Sheep. Biomimetics
(one site/animal; negative control (Neg. Ctrl)) and left intact at twelve sites (one site/animal; experi-
2024, 9, 568. https://doi.org/ mental group (Exp.)). All osteotomies were created using the osseodensification drilling protocol.
10.3390/biomimetics9090568 Each osteotomy received an endosteal implant and was evaluated after 3 or 12 weeks of healing (n = 6
animals/time). Histology revealed increased woven and lamellar bone surrounding the implants in
Academic Editor: Bo Su
the Exp. group relative to the Neg. Ctrl group. The Exp. group demonstrated the presence of bone
Received: 13 August 2024 fragments, which acted as nucleating sites, thereby enhancing the bone formation and remodeling
Revised: 10 September 2024 processes. Bone-to-implant contact (%BIC) and bone area fractional occupancy (%BAFO) were sig-
Accepted: 16 September 2024 nificantly higher in the Exp. group relative to the Neg. Ctrl group both at 3 weeks (p = 0.009 and
Published: 19 September 2024 p = 0.043) and 12 weeks (p = 0.010 and p = 0.008). Osseodensification instrumentation in the presence
of trabecular bone significantly improved osseointegration. However, no negative influences such as
necrosis, inflammation, microfractures, or dehiscence were observed in the absence/limited presence
of trabeculae.
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Keywords: osseodensification; implants; additive instrumentation; osteotomy; trabecular bone
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Biomimetics 2024, 9, 568. https://doi.org/10.3390/biomimetics9090568 https://www.mdpi.com/journal/biomimetics

Biomimetics 2024, 9, 568 2 of 11

1. Introduction
Tooth loss occurring from iatrogenic, traumatic, or therapeutic causes often leads to
edentulism [1], therefore necessitating the utilization of implants and their subsequent
anchorage for long-term rehabilitation [2,3]. While effective, one of the most common
complications is implant screw loosening, estimated to affect up to ~12% of patients [4–6],
which is due to a variety of clinical scenarios (i.e., technical errors during positioning,
screw length, inappropriate mechanical transmission of stress on the device, and/or poor
bone quality) [7–9]. Furthermore, the frequency of early implant failure increases with
additional factors such as smoking, the absence of post-operative antibiotic therapy, bone
augmentation, and implant dimensions. Procedures to address implant failure can be
financially prohibitive endeavors and potentially difficult technical procedures due to
the scarred, distorted anatomy and, in certain circumstances, the need to use specialized
instruments [10,11].
Several different approaches, such as the modification of screw length, diameter, or
insertion trajectory; fenestration design; and osteotomy preparation techniques, have been
employed to resolve implant failure [12,13]. For successful implant placement, adequate
bone compression around the screw upon immediate insertion and long-term screw fixation
(primary and secondary stability, respectively) are paramount [14]. Primary stability is
essential to long-term success as it prevents the micromotion of the implant during the early
stages of the healing process. The degree of primary stability can be influenced by implant
design, osteotomy size, bone density, and/or patient comorbidities [15]. Increased levels of
primary stability at the time of implant insertion have been shown to elicit rapid secondary
stability [16]. This necessitates maximizing primary stability at the time of endosteal
implant insertion to thereby increase the probabilities of long-term implant fixation.
A potential option for maximizing primary stability is increasing bone-to-implant
contact [17]. Instrumentation (e.g., drilling technique) is a primary aspect to be considered
when high primary stability is desired. Several instrumentation modalities have been
suggested to increase primary stability, particularly in low-density bone [18–20]. How-
ever, the introduction and implementation of additive instrumentation techniques (e.g.,
osseodensification) to improve osseointegration has repeatedly demonstrated favorable
results in attempts to minimize implant loosening by maximizing primary stability and
osteointegration [21,22]. To elaborate, osseodensification is a form of ‘additive instrumen-
tation’ whereby the burr compacts the bone fragments into the osteotomy wall [23] and
the preserved bone chips (e.g., autografts) act as nucleating surfaces at the bone-to-implant
interface, facilitating bone formation and osseointegration [24,25].
The efficacy and viability of osseodensification instrumentation relative to conven-
tional subtractive techniques within uncompromised (native) trabecular bone have been
established [22]. Osseodensification sub-instrumentation, the use of additive instrumenta-
tion in the context of different macro-geometries, and the surface parameters of implants
have also been explored [26]. However, a systematic review in 2020 by Padhye et al. in-
dicated that while osseodensification may be particularly useful in areas with relatively
high amounts of cancellous bone, its use in areas with limited trabecular bone (primar-
ily corticated bone) warranted evaluation due to a dearth of published data [24]. Yet,
pre-clinical and clinical studies in the literature focusing on the use of osseodensification
instrumentation have largely not accounted for limited trabecular bone volume at the
osteotomy site [27].
In addition, a study by Koutouzis et al. suggested that osseodensification in sites with
a limited volume of trabecular bone may produce a higher risk of bone overstraining and
microfractures, although this has yet to be established through pre-clinical or clinical evalu-
ation [28]. Building on these findings and assumptions, the objective of the current study
was to evaluate the bone regeneration outcomes in the immediate vicinity of endosteal
implants inserted into osteotomy sites prepared using osseodensification drilling in the
limited presence of underlying trabecular bone.
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Biomimetics 2024, 9, 568 3 of 11

2. 2. Methods
Methods
2.1.
2.1. Pre-Clinical
Pre-Clinical Experiments
Experiments
Upon
Uponapproval
approvalfrom fromÉcole
ÉcoleNationale
NationaleVétérinaire
Vétérinaired’Alfort
d’Alfort(Maisons-Alfort,
(Maisons-Alfort,Ile-de- Ile-de-
France,
France,France)
France)Institutional
InstitutionalAnimal
AnimalCare Careand
and UseUse Committee (IACUC), 12
Committee (IACUC), 12 adult
adultsheep
sheep[n
[n==66perpertime
timein invivo
vivo(3(3 and
and 1212 weeks)] were obtained
obtained and andallowed
allowedto toacclimate
acclimatefor forapprox-
approx-
imately one week. All surgical procedures were performed under
imately one week. All surgical procedures were performed under an aseptic environment an aseptic environment
andand general
general anesthesia.
anesthesia. Each
Each animal
animal was
wasinjected
injected withwithsodium
sodium pentathol
pentathol (15–20
(15–20 mg/kg)
mg/kg)
inina Normasol
a Normasol solution
solution in in
thethe
jugular
jugularvein.
vein.Anesthesia
Anesthesiawas wasmaintained
maintainedwith withisoflurane
isoflurane
(1.5–3%)
(1.5–3%) ininO2O /N2/N2O2O(50/50).
(50/50). Concurrently,
Concurrently,ECG, ECG,SpO SpO2 ,2,and
andfinal
finaltidal
tidalCO CO2 were
2 were used
usedtoto
track
trackvital
vitalsigns.
signs.TheTheilium
iliumwas wasselected
selectedasasthe
thesite
sitefor
forosteotomy
osteotomyand andimplant
implantplacement.
placement.
AtAt the
thetime
time ofofsurgery,
surgery, the site
the was
site wasshaved
shaved andand prepared
prepared with anan
with iodine
iodine solution,
solution,followed
followed
byby ananincision of ~10cm in the anteroposterior direction over the iliac
incision of ~10cm in the anteroposterior direction over the iliac crest. Subsequently,crest. Subsequently,
iliac bone
iliac bone waswas exposed
exposed andand 2 osteotomies
2 osteotomies werewere prepared
prepared asas
follows:
follows: (1)(1)
with
withtrabecular
trabecular
bone (Exp.) and (2) limited bone (Neg. Ctrl) in the trabecular space;
bone (Exp.) and (2) limited bone (Neg. Ctrl) in the trabecular space; i.e., trabecular i.e., trabecular bone
bone
waswas surgically
surgically removed/deburred
removed/deburred from
fromthe
theiliac crest
iliac crest between
between the
thecortical
cortical plates atat
plates the
thesite
site
ofofsurgery,
surgery, leaving the cortical bone thickness intact prior to osseodensification drillingaat
leaving the cortical bone thickness intact prior to osseodensification drilling at
total of twenty-four
a total of twenty-four sitessites
(Figure 1). 1).
(Figure

Figure 1. Schematic of the 2 groups (Neg. Ctrl and Exp.) used in this study. Image generated on
Figure 1. Schematic of the 2 groups (Neg. Ctrl and Exp.) used in this study. Image generated on
Biorender.com.
Biorender.com.
Osseodensification drilling (Figure 2) was performed in a counterclockwise fashion
Osseodensification drilling (Figure 2) was performed in a counterclockwise fashion
using the Densah® multifluted tapered bur (Versah LLC., Jackson, MI, USA) 2.0 mm pilot
using the Densah
drill, followed
® multifluted tapered bur (Versah LLC., Jackson, MI, USA) 2.0 mm pilot
by the 2.8 mm and 3.8 mm burs at 1100 rpm under continuous saline
drill, followed
irrigation, by the each
after which 2.8 mm and 3.8 mm
osteotomy burs at
received 1100 rpm under
a Ti-6Al-4V screw continuous saline irri-
root form endosteal
gation, after which each osteotomy received a Ti-6Al-4V screw root form
implant (Emfils, Itu, Brazil)—4 mm in diameter, 10 mm in length, and with a machined endosteal im-
plant (Emfils, Itu, Brazil)—4 mm in diameter, 10 mm in length, and with a machined
(regular) surface with no additional surface treatments. All implants were torqued into the (reg-
ular) surface
osteotomy with
as per the no additional surface
manufacturer’s treatments.
specifications All implants
(85 N.cm ± 10%).were torqued into the
osteotomy as per the manufacturer’s specifications (85 N.cm ± 10%).
Biomimetics
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FOR PEER REVIEW 4 of
4 of1111

(A)Computer-Aided
Computer-Aided Design images ® multifluted tapered burs (Versah LLC,
Figure2.2.(A)
Figure Design images of of Densah
Densah ® multifluted tapered burs (Versah LLC,
MI,
MI,USA).
USA).Reproduced
Reproduced with permission
with permission from [26],
from copyright
[26], 2018
copyright Orthopaedic
2018 Research
Orthopaedic Society.
Research Society.
Published by Wiley Periodicals, Inc. (B) Schematic picture of osseodensification drilling method
Published by Wiley Periodicals, Inc. (B) Schematic picture of osseodensification drilling method
(Image
(Imagecourtesy
courtesyofofVersah
VersahLLC,
LLC,MI,
MI,USA).
USA).Reproduced
Reproduced with
withpermission from
permission from[29], copyright
[29], 2020
copyright 2020
Orthopaedic Research Society. Published by Wiley Periodicals
Orthopaedic Research Society. Published by Wiley Periodicals LLC. LLC.

Subjects
Subjects were randomlyallocated
were randomly allocatedtoto
one one of two
of two healing
healing times,times, 3 weeks
3 or 12 or 12 weeks (n =
(n = 6/time).
6/time). Surgical sites were sutured using Vicryl 2-0 for muscle and
Surgical sites were sutured using Vicryl 2-0 for muscle and nylon 2-0 for skin. Cefazolinnylon 2-0 for skin.
Cefazolin
(500 mg) was(500administered
mg) was administered pre-operatively
pre-operatively and post-operatively
and post-operatively via intravenousvia injections
intrave-
nous injections
to reduce to reduce the
the appearance appearance of post-operative
of post-operative complications.complications.
Animals were Animals
providedwere food
provided
and water food and water
ad libitum. ad libitum.
Three Three
and twelve and after
weeks twelvetheweeks after theintervention,
first surgical first surgicalanimals
inter-
vention, animals were
were euthanized euthanized
according to the according to the approved
approved protocol and samples protocol
wereand samplesenwere
harvested bloc.
harvested en bloc.
2.2. Histomorphometric Analysis
2.2. Histomorphometric
The bone–implant Analysis
blocks were gradually dehydrated in a series of alcohol solutions
ranging from 70% to 100%
The bone–implant blocks ethanol.
were Following dehydration,inthe
gradually dehydrated samples
a series were immersed
of alcohol solutionsin
a clearing solution (methyl salicylate) and subsequently embedded
ranging from 70% to 100% ethanol. Following dehydration, the samples were immersed in a methacrylate-based
inresin. The embedded
a clearing samples
solution (methyl were cutand
salicylate) intosubsequently
slices (~300 µm) with a in
embedded precision diamond
a methacrylate-
wafering saw (Isomet ® Low Speed, Buehler Ltd., Lake Bluff, IL, USA). The slices were
based resin. The embedded samples were cut into slices (~300 µm) with a precision dia-
generated
mond transversally
wafering saw (Isomet(relative
® Lowto the long
Speed, axis of
Buehler theLake
Ltd., implant),
Bluff,such that the
IL, USA). Theimplant
slices
cross-section was visible.
were generated transversally (relative to the long axis of the implant), such that the im-
Sections werewas
plant cross-section glued to acrylic slides with a cyanoacrylate-based adhesive (Loctite 408,
visible.
Henkel AG, Dusseldorf, Germany),
Sections were glued to acrylic slides and a 24-h
with setting time was allowedadhesive
a cyanoacrylate-based prior to grinding
(Loctite
and polishing. The sections were then reduced to a final
408, Henkel AG, Dusseldorf, Germany), and a 24-h setting time was allowed thickness of ~80 µm by means
prior to
of a series of silicon carbide (SiC) abrasive papers (400, 600, 800, and
grinding and polishing. The sections were then reduced to a final thickness of ~80 µm by 1200 grit; Buehler
Ltd., Lake Bluff, IL, USA) using a grinding/polishing machine (Metaserv 3000, Buehler
means of a series of silicon carbide (SiC) abrasive papers (400, 600, 800, and 1200 grit;
Ltd., Lake Bluff, IL, USA) under copious water irrigation. Subsequently, the samples were
Buehler Ltd., Lake Bluff, IL, USA) using a grinding/polishing machine (Metaserv 3000,
stained with Stevenel’s Blue and Van Gieson’s Picro Fuchsin (SVG) and digitally scanned
Buehler Ltd., Lake Bluff, IL, USA) under copious water irrigation. Subsequently, the sam-
via an automated slide scanning system (Aperio CS2, Vista, CA, USA) and specialized
ples were stained with Stevenel’s Blue and Van Gieson’s Picro Fuchsin (SVG) and digitally
computer software (Aperio ImageScope 12.4.6, Vista, CA, USA). Stevenel’s Blue stained
scanned via an automated slide scanning system (Aperio CS2, Vista, CA, USA) and spe-
cells and extracellular structures in a subtle gradation of blue tones. The counterstain, Van
cialized computer software (Aperio ImageScope 12.4.6, Vista, CA, USA). Stevenel’s Blue
Gieson’s Picro Fuchsin, stained collagen fibers green or green-blue; bone in red, orange,
stained cells and extracellular structures in a subtle gradation of blue tones. The counter-
or purple; and muscle fibers in blue to blue-green. Bone-to-implant contact (%BIC) in the
stain, Van Gieson’s Picro Fuchsin, stained collagen fibers green or green-blue; bone in red,
cancellous layers was quantified (one slide from each implant/osteotomy was chosen)
orange, or purple; and muscle fibers in blue to blue-green. Bone-to-implant contact (%BIC)
in the cancellous layers was quantified (one slide from each implant/osteotomy was
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Biomimetics 2024, 9, 568 5 of 11

chosen) using image analysis software (ImageJ 1.54h, NIH, Bethesda, MD, USA) as per the
following equation
using image analysis(and illustrated
software in 1.54h,
(ImageJ FigureNIH,
3A,B):
Bethesda, MD, USA) as per the following
equation (and illustrated in Figure 3A,B):
𝑇ℎ𝑒 𝑝𝑒𝑟𝑖𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 𝑡ℎ𝑒 𝑖𝑚𝑝𝑙𝑎𝑛𝑡 𝑠𝑢𝑟𝑓𝑎𝑐𝑒 𝑐𝑜𝑣𝑒𝑟𝑒𝑑 𝑤𝑖𝑡ℎ 𝑛𝑒𝑤 𝑏𝑜𝑛𝑒 100
%𝐵𝐼𝐶
𝑇ℎ𝑒
The𝑡𝑜𝑡𝑎𝑙 𝑝𝑒𝑟𝑖𝑚𝑒𝑡𝑒𝑟
perimeter 𝑜𝑓 𝑡ℎ𝑒 𝑖𝑚𝑝𝑙𝑎𝑛𝑡
o f the implant sur f ace covered with new bone × 100
%BIC =
On the other hand, bone area The total perimeter
fractional occupancyo f the(%BAFO)
implant was used to quantify
the osteogenic parameters
On the other hand, bonein the
arearegion of interest
fractional (ROI).(%BAFO)
occupancy The ROI is wasdefined as quantify
used to the area
between the green highlights in Figure 3C. To elaborate, the green circle
the osteogenic parameters in the region of interest (ROI). The ROI is defined as the is up to 1 area
mm
away from the implant surface. %BAFO was quantified (one slide from each implant/os-
between the green highlights in Figure 3C. To elaborate, the green circle is up to 1 mm away
teotomy
from the was chosen)
implant in the%BAFO
surface. cancellouswaslayers using(one
quantified image analysis
slide software
from each (ImageJ 1.54h,
implant/osteotomy
NIH, Bethesda,
was chosen) MD,cancellous
in the USA) according
layers to the following
using image analysisequation (and illustrated
software in Figure
(ImageJ 1.54h, NIH,
3C–E):
Bethesda, MD, USA) according to the following equation (and illustrated in Figure 3C–E):
𝐵𝑜𝑛𝑒 𝑎𝑟𝑒𝑎 𝑤𝑖𝑡ℎ𝑖𝑛 𝑡ℎ𝑒 𝑅𝑂𝐼 100
%𝐵𝐴𝐹𝑂 Bone area within the ROI × 100
%BAFO = 𝑇𝑜𝑡𝑎𝑙 𝑎𝑟𝑒𝑎 𝑒𝑛𝑐𝑜𝑚𝑝𝑎𝑠𝑠𝑒𝑑 𝑤𝑖𝑡ℎ𝑖𝑛 𝑡ℎ𝑒 𝑅𝑂𝐼
Total area encompassed within the ROI

Figure 3. Representative
Figure 3. Representative histomicrographs
histomicrographsshowing
showing(A) (A)the
the perimeter
perimeter of of
thethe implant
implant surface
surface cov-
covered
ered with new bone (yellow lines), (B) the total transverse perimeter of the implant (blue lines), (C)
with new bone (yellow lines), (B) the total transverse perimeter of the implant (blue lines), (C) the
the region of interest (ROI) in green highlights, (D) the total area encompassed within the ROI, and
region of interest (ROI) in green highlights, (D) the total area encompassed within the ROI, and
(E) the bone area within the ROI. Bone is colored red due to SVG staining.
(E) the bone area within the ROI. Bone is colored red due to SVG staining.
The choice of section plane has been suggested to have an influence on the histomor-
The choice of section plane has been suggested to have an influence on the histomor-
phometric findings [30]. However, it has been established that the same cutting/sectioning
phometric findings [30]. However, it has been established that the same cutting/sectioning
plane can be taken into account to quantify the bone growth around the implant to stand-
plane can be taken into account to quantify the bone growth around the implant to stan-
ardize the results [30]. As such, to ensure that %BIC and %BAFO values are comparable
dardize the results [30]. As such, to ensure that %BIC and %BAFO values are comparable
between implants and/or test subjects, a standardized selection of the sectioning plane
between implants and/or test subjects, a standardized selection of the sectioning plane was
was performed.
performed. OnceOnce the sectioning
the sectioning planeplane (transverse
(transverse to implant
to the the implant longitudinal
longitudinal axis)axis)
was
was
defined to be located 5 mm from the apex of the implant (illustrated in Figure 4), it4),
defined to be located 5 mm from the apex of the implant (illustrated in Figure it
was
was kept consistent among all the slides analyzed to minimize
kept consistent among all the slides analyzed to minimize bias. bias.
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Figure 4. Schematic illustration of the histological sectioning plane (in dashed red lines) chosen
Figure 4. Schematic illustration of the histological sectioning plane (in dashed red lines) chosen for
for histomorphometric analyses. CB = Cortical Bone; TB = Trabecular Bone. Image generated on
histomorphometric analyses. CB = Cortical Bone; TB = Trabecular Bone. Image generated on Bioren-
Biorender.com.
der.com.
2.3. Statistical Analysis
2.3. Statistical Analysis
Statistical analysis was performed using IBM SPSS (v29, IBM Corp., Armonk, NY,
USA),Statistical analysis was performed
with histomorphometry usingasIBM
data presented mean SPSS (v29,
values IBM
with 95%Corp., Armonk,
confidence NY,
interval
USA), with histomorphometry data presented as mean values with 95% confidence
values (mean ± 95%CI). The assessment of the normality of the data is a prerequisite for inter-
val values (mean
parametric ± 95%CI).
statistical The underlying
tests given assessmentassumptions.
of the normality of thedue
As such, datato is a prerequisite
the sample size,
for parametric statistical tests given underlying assumptions. As such,
the Shapiro–Wilk test was performed to confirm data normality (p > 0.05), prior due to the sample
to the use
size, the Shapiro – Wilk test was performed to confirm data normality (p
of an appropriate statistical analysis. The values of %BIC and %BAFO were analyzed with > 0.05), prior to
athe use of
linear an appropriate
mixed statistical
model (analogous to analysis. The valuesand
logistic regression of %BIC and %BAFO
optimized were
for nested ana-
within
lyzed with
subject a linear mixed
observations) and fixedmodel (analogous
factors of surgicaltoinstrumentation
logistic regression and (Neg.
method optimized for
Ctrl and
nestedand
Exp.) within
timesubject
in vivoobservations)
(3 and 12 weeks).and fixed factors of surgical instrumentation method
(Neg. Ctrl and Exp.) and time in vivo (3 and 12 weeks).
3. Results
3. Results
During the immediate post-operative evaluation, no surgical site revealed any sign
of inflammation or infection,post-operative
During the immediate and there wasevaluation,
no evidence noofsurgical
implantsite
failure at theany
revealed time of
sign
necropsy.
of inflammation or infection, and there was no evidence of implant failure at the time of
necropsy.
3.1. Qualitative Histologic Findings
Bone formation
3.1. Qualitative around
Histologic the endosteal implants was qualitatively analyzed at the
Findings
two healing
Bone formation around theweeks,
time points. At 3 the implants
endosteal Neg. Ctrlwas
group (Figure 5A)
qualitatively demonstrated
analyzed a
at the two
limited presence of newly formed woven bone in the immediate periphery of the implant.
healing time points. At 3 weeks, the Neg. Ctrl group (Figure 5A) demonstrated a limited
Relative to the Neg. Ctrl group, the Exp. group (Figure 5B) presented with increased
presence of newly formed woven bone in the immediate periphery of the implant. Rela-
degrees of woven bone surrounding the implant, suggesting greater primary stability at
tive to the Neg. Ctrl group, the Exp. group (Figure 5B) presented with increased degrees
the early healing time point. Additionally, the osseodensification instrumentation in the
of woven bone surrounding the implant, suggesting greater primary stability at the early
presence of the trabecular network resulted in the compaction of a larger number of bone
healing time point. Additionally, the osseodensification instrumentation in the presence
chips towards the osteotomy walls (blue arrows, Figure 5B) surrounding the immediate
of the trabecular network resulted in the compaction of a larger number of bone chips
vicinity of the implant. Bone chips were attached and embedded into the osteotomy wall
towards the osteotomy walls (blue arrows, Figure 5B) surrounding the immediate vicinity
and the implant surface.
of the implant. Bone chips were attached and embedded into the osteotomy wall and the
implant surface.
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Figure 5. Representative histomicrographs of the Neg. Ctrl and Exp. groups at (A,B) 3 and (C,D)
Figure 5. Representative histomicrographs of the Neg. Ctrl and Exp. groups at (A,B) 3 and (C,D)
12 weeks, respectively. Blue arrows represent the bone fragments compacted around the implant
12 weeks, respectively. Blue arrows represent the bone fragments compacted around the implant as a
as a result of osseodensification instrumentation. Green arrows point toward bone remodeling
resultinofthe
sites osseodensification instrumentation.
immediate vicinity of the implantGreen
in thearrows point toward bone remodeling sites in the
Exp. group.
immediate vicinity of the implant in the Exp. group.
At the extended healing time, 12 weeks, the Neg. Ctrl group (Figure 5C) revealed
At the extended healing time, 12 weeks, the Neg. Ctrl group (Figure 5C) revealed
increased bone formation with indications of lamellar reorganization relative to the 3-
increased bone formation with indications of lamellar reorganization relative to the 3-week
week time point. The Exp. group (Figure 5D) presented higher degree of new bone for-
time point. The Exp. group (Figure 5D) presented higher degree of new bone formation
mation surrounding the implant. Furthermore, bone formation in the Exp. group was
surrounding the implant. Furthermore, bone formation in the Exp. group was more
more pronounced at this advanced healing time point relative to 3 weeks, with the new
pronounced at this advanced healing time point relative to 3 weeks, with the new bone
bone surrounding the implant appearing lamellar and progressing toward advanced de-
surrounding the implant appearing lamellar and progressing toward advanced degrees of
grees of remodeling (green arrows, Figure 5D). Similar to the early healing time point,
remodeling (green arrows, Figure 5D). Similar to the early healing time point, bone chips
bone chips as a result of the osseodensification process in the Exp. group were found to
as a result of the osseodensification process in the Exp. group were found to be embedded
be embedded into the osteotomy wall and the implant surface (blue arrows, Figure 5D),
into the osteotomy wall and the implant surface (blue arrows, Figure 5D), seemingly acting
seemingly acting as autologous grafting particles owing to their encapsulation by the re-
as autologous grafting particles owing to their encapsulation by the remodeling bone.
modeling bone.
3.2. Histomorphometric Analysis
3.2. Histomorphometric Analysis
The quantitative evaluation of bone-to-implant contact (%BIC) independent of time
Theyielded
in vivo quantitative evaluation
significantly higherof results
bone-to-implant
in the Exp.contact (%BIC) independent
group (30.12% ± 7.5) relativeoftotime
the
in vivo yielded significantly higher results in the Exp. group (30.12%
Neg. Ctrl group (10.94% ± 7.5) (p = 0.001) (Figure 6A). Subsequent analyses ± 7.5) relative
of %BIC toasthea
Neg. Ctrlof
function group (10.94%
drilling ± 7.5)and
technique (p = time
0.001)
in(Figure 6A). Subsequent
vivo detected analyses of at
statistical differences %BIC
3 weeksas a
function
(p = 0.009)of drilling technique
and 12 weeks (p =and timewhere
0.010), in vivothedetected statistical
Exp. group differences
[30.87% ± 11.2at(33weeks)
weeks
(p
and= 0.009)
29.36%and 12 weeks
± 11.2 (p = 0.010),
(12 weeks)] where
yielded the Exp.
higher group
values [30.87%
relative ± 11.2
to the (3 weeks)
Neg. Ctrl groupand
29.36%
[11.42%±±11.211.2(12
(3 weeks)] yielded
weeks) and 10.47%higher values
± 11.2 relative (Figure
(12 weeks)] to the Neg.
6B). Ctrl group [11.42% ±
11.2 (3 weeks)
The and 10.47%
evaluation ± 11.2
of bone area(12 weeks)]occupancy
fractional (Figure 6B).(%BAFO) independent of healing
time was significantly higher in the Exp. group (16.74% ± 2.9) in comparison to the Neg.
Ctrl group (11.38% ± 2.9) (p = 0.003) (Figure 7A). Additionally, %BAFO results as a function
of instrumentation technique (Neg. Ctrl vs. Exp.) and time in vivo (3 vs. 12 weeks) revealed
statistical differences. For instance, %BAFO was higher in the Exp. group (15.36% ± 3.9)
relative to the Neg. Ctrl group (11.48% ± 3.9) at 3 weeks (p = 0.043). A similar trend was
observed at the advanced healing time point, where the Exp. group yielded higher %BAFO
(18.12% ± 3.9) in comparison to the Neg. Ctrl group (11.28% ± 3.9) (p = 0.008) (Figure 7B).
Biomimetics 2024, 9, x FOR PEER REVIEW 8 of 11
Biomimetics 2024, 9, 568 8 of 11

Figure 6. Histomorphometric data. (A) %BIC collapsed over time (3 and 12 weeks), and (B) %BIC
as a function of experimental group and time. p < 0.05 is statistically homogeneous.

The evaluation of bone area fractional occupancy (%BAFO) independent of healing

time was significantly higher in the Exp. group (16.74% ± 2.9) in comparison to the Neg.
Ctrl group (11.38% ± 2.9) (p = 0.003) (Figure 7A). Additionally, %BAFO results as a function
of instrumentation technique (Neg. Ctrl vs. Exp.) and time in vivo (3 vs. 12 weeks) re-
vealed statistical differences. For instance, %BAFO was higher in the Exp. group (15.36%
± 3.9) relative to the Neg. Ctrl group (11.48% ± 3.9) at 3 weeks (p = 0.043). A similar trend
was observed at the advanced healing time point, where the Exp. group yielded higher
%BAFO (18.12% ± 3.9) in comparison
Histomorphometric data. (A) tocollapsed
(A) %BIC
%BIC the Neg. Ctrl group (11.28% ± 3.9) (p = 0.008)
Figure 6.
Figure 6. Histomorphometric data. collapsed over
over time
time (3
(3 and
and 12
12 weeks),
weeks), and
and (B)
(B) %BIC
%BICas
(Figure 7B).
a function of experimental group and time. p < 0.05 is statistically homogeneous.
as a function of experimental group and time. p < 0.05 is statistically homogeneous.

The evaluation of bone area fractional occupancy (%BAFO) independent of healing

time was significantly higher in the Exp. group (16.74% ± 2.9) in comparison to the Neg.
Ctrl group (11.38% ± 2.9) (p = 0.003) (Figure 7A). Additionally, %BAFO results as a function
of instrumentation technique (Neg. Ctrl vs. Exp.) and time in vivo (3 vs. 12 weeks) re-
vealed statistical differences. For instance, %BAFO was higher in the Exp. group (15.36%
± 3.9) relative to the Neg. Ctrl group (11.48% ± 3.9) at 3 weeks (p = 0.043). A similar trend
was observed at the advanced healing time point, where the Exp. group yielded higher
%BAFO (18.12% ± 3.9) in comparison to the Neg. Ctrl group (11.28% ± 3.9) (p = 0.008)
(Figure 7B).

Figure 7. Histomorphometric
Figure 7. Histomorphometricdata.data.
(A)(A) %BAFO
%BAFO collapsed
collapsed overover
time time
(3 and(312and 12 weeks),
weeks), and (B) and (B)
%BAFO
%BAFO as a function
as a function of experimental
of experimental group
group and time.and
p <time. p <statistically
0.05 is 0.05 is statistically homogeneous.
homogeneous.

4. Discussion
Discussion
Conventional, subtractive
Conventional, subtractive drilling
drilling instrumentation
instrumentation pertaining
pertaining to to implant
implant fixation
fixation has
has
been utilized and is extensively available in the literature. Nevertheless, its
been utilized and is extensively available in the literature. Nevertheless, its limitation (i.e., limitation (i.e.,
excavating bone
excavating bone atat the
the site
siteof
ofthe
theosteotomy)
osteotomy)has hasbeen
beenshown
shown totonegatively
negatively impact
impact bone
bonere-
generation, with
regeneration, withremodeling
remodeling ultimately
ultimatelyresulting
resultingin the lossloss
in the of viable bone
of viable bonefragments
fragments at the
at
bone–implant interface which have the capacity to bridge the gap
the bone–implant interface which have the capacity to bridge the gap between the osteot-between the osteotomy
wallswalls
omy and the
andimplant
the implantsurface, thus thus
surface, necessitating
necessitatingthe usetheofuse
additive instrumentation
of additive instrumentation tech-
niques such
techniques as osseodensification.
such as osseodensification. Studies pertaining
Studies to osseodensification
pertaining instrumentation
to osseodensification instru-
Figure 7. Histomorphometric data. (A) %BAFO collapsed over time (3 and 12 weeks), and (B)
have highlighted
mentation have its efficacious bone
highlighted its regenerative
efficacious capabilities
bone [22,26].capabilities
regenerative However, the afore-
[22,26].
%BAFO as a function of experimental group and time. p < 0.05 is statistically homogeneous.
mentioned studies that outline the use of osseodensification in pre-clinical settings have
not elucidated its effect on bone regeneration around endosteal implants placed in areas
4. Discussion
with limited trabecular volume.
Conventional,
In this context,subtractive
the currentdrilling
study instrumentation
examined the effect pertaining to implant fixation
of osseodensification has
drilling
been utilized and is extensively available in the literature. Nevertheless,
with and without trabecular bone in a large translational model. Utilizing the sheep its limitation (i.e.,
excavating bonestudy
model for this at theand siteselecting
of the osteotomy)
the ilium hasduebeento itsshown to negatively
low-density impact bone
bone configuration
regeneration,
allowed for the with remodeling groups
experimental ultimately
to beresulting
nested in the loss
within of viable
each animalboneowing fragments at
to its size,
the bone–implant
thereby maximizing interface which
statistical havewhile
power the capacity
minimizingto bridge the gap between
the number of animals theused
osteot-
for
omy walls and the Additionally,
experimentation. implant surface, thuslow-bone-density
using necessitating the use sitesofsuch
additive
as the instrumentation
hip effectively
techniques such as osseodensification. Studies
simulated the low-bone-density settings seen clinically. pertaining to osseodensification instru-
mentation have highlighted its efficacious bone regenerative capabilities [22,26].
Biomimetics 2024, 9, 568 9 of 11

The qualitative result of the current study strongly indicates that osseodensification
drilling did not have any negative influence (i.e., necrosis, inflammation, or dehiscence)
on bone healing. The quantitative histomorphometric analyses confirmed that healing
outcomes were significantly greater in the presence of trabecular bone volume at both the
early (3-week) and advanced (12-week) healing time points. Pertaining to the Exp. Group,
contained within the trabecular bone, between the layers of cortical bone, is a porous
network of bone cells and marrow [31]. This sponge-like cellular network has been shown
to result in an increased surface area of bone cells relative to cortical bone [32], which not
only elicits greater bone formation but also facilitates osseodensification instrumentation.
The osseodensification burs possess a large negative rake angle and act as non-cutting
edges to allow for the compaction of bone fragments, thereby increasing bone density at
the site of the osteotomy. Das et al. [33] described the mechanism of the osseodensification
drilling technique in which counterclockwise rotation compacts bone particles into the
trabecular wall. Once the bone particles are coated to the osteotomy walls, they promote
increased bone density at the bone–screw surface while also promoting primary stability
and, as a result, increased osseointegration [34]. This was confirmed through the qualitative
histomicrographs of the Exp. group at both time points, seen as the preservation of the bone
bulk through the enhancement in bone density by the lateral compaction or displacement
of autografting bone particles at the walls of the osteotomy. Osseodensification was
seen to preserve the bone-chip autografts which acted as nucleating surfaces at the bone–
implant interface, ultimately facilitating osseointegration, as seen previously [26]. Such
a phenomenon was not observed in the Neg. Ctrl group, largely owing to the lack of
trabecular bone volume, highlighting the requirement of trabecular bone at the site of the
osteotomy to achieve sufficient osseodensification using Densah® multifluted tapered burs.
On the other hand, pertaining to the comparison of osseodensification instrumentation
to conventional subtractive (osteotomy) techniques, have been reported by Buchter et al.
that the latter hampers bone remodeling and causes ultrastructural microdamage, and
that the biomechanical stability may be significantly decreased shortly after implant place-
ment [35]. This could be attributed to microcracking at the osteotomy sites due to strain
that exceeds bone’s elasticity. Furthermore, in another study by Alifarag et al., histologic
micrographs around the osseodensification instrumented implants demonstrated a lower
incidence of microcracking due to compression relative to conventional drilling [26]. While
the current study qualitatively and quantitatively highlighted the dependence of osseoden-
sification instrumentation on presence of trabecular volume, it also demonstrated positive
healing outcomes in its use with limited trabeculae, with no indications of microdamage
at either healing time (3 or 12 weeks). This warrants future studies that directly compare
osseodensification instrumentation to conventional subtractive techniques, specifically in
the absence of trabecular bone volume at the osteotomy site prior to implant placement.
In conclusion, the histomorphometric evaluation of osseodensification instrumentation
in the presence of trabecular bone volume (Exp.) exhibited improved healing outcomes
relative to sites with limited trabeculae (Neg. Ctrl). Nonetheless, osseodensification drilling
had no negative influence such as necrosis, inflammation, or dehiscence on bone healing,
with no indications of microdamage or microfractures. In a systematic review from 2024,
Kalra et al. reported that human clinical research pertaining to osseodensification usage
has produced predictable and positive effects [36]. However, despite a number of research
investigations, the existing literature mostly consists of studies on animals or clinical cases
with short-term follow-ups [21]. One contributing factor to this could be attributed to
the novelty of the drills used for osseodensification, which are still not widespread in
standard clinical practice [37]. However, this demand is expected to rise in tandem with
upcoming in vivo research which could focus on evaluating the effect of osseodensification
instrumentation on bone healing through long-term follow-ups [37].
Biomimetics 2024, 9, 568 10 of 11

Author Contributions: Conceptualization, L.W. and P.G.C.; methodology, E.A.B., L.W. and P.G.C.;
software, A.W., E.T.P.B., V.V.N. and M.V.P.; investigation, Z.S., S.W., J.E.H., A.W., A.C., S.K.J., E.T.P.B.,
N.A.M., V.V.N., M.V.P., E.A.B., L.W. and P.G.C.; resources, L.W. and P.G.C.; data curation, Z.S., S.W.,
J.E.H., N.A.M., A.W., E.T.P.B., V.V.N., A.C., S.K.J. and M.V.P.; writing—original draft preparation, Z.S.,
S.W., J.E.H. and N.A.M.; writing—review and editing, V.V.N., M.V.P., E.A.B., L.W. and P.G.C.; visual-
ization, A.W., E.T.P.B., V.V.N. and L.W.; supervision, E.A.B., L.W. and P.G.C.; project administration,
L.W. and P.G.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board of École Nationale Vétérinaire d’Alfort (Maisons-Alfort, Ile-de-France, France) with
file number: 13-011; notice number: 05/14/13-3; and date of approval: 14 May 2013.
Data Availability Statement: The original contributions presented in the study are included in the
article, and further inquiries can be directed to the corresponding author.
Acknowledgments: The authors would like to express their gratitude to Versah LLC., MI, USA.
Conflicts of Interest: The authors declare no conflicts of interest.

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