Post on 11-Jan-2022
Implant-guided vertical bone growth inthe mini-pig
Martin FreilichBo WenDavid ShaferPeter SchleierMichel DardDavid PendrysDenise OrtizLiisa Kuhn
Authors’ affiliations:Martin Freilich, Liisa Kuhn, Bo Wen, Denise Ortiz,Department of Reconstructive Sciences, Center forBiomaterials, School of Dental Medicine, University ofConnecticut, Farmington, CT, USADavid Shafer, Department of Craniofacial Sciences,Division of Oral & Maxillofacial Surgery, School ofDental Medicine, University of Connecticut,Farmington, Ct, USADavid Pendrys, Department of ReconstructiveSciences, School of Dental Medicine, University ofConnecticut, Farmington, CT, USAPeter Schleier, Oralfacial Surgery Section, Clinic forSpecial Medicine, Stavanger, NorwayMichel Dard, Institut Straumann, Basel, Switzerland
Corresponding author:Dr Martin FreilichDepartment of Reconstructive SciencesSchool of Dental MedicineUniversity of Connecticut263 Farmington AvenueFarmingtonCT USATel.: 860 679 2649Fax: 860 679 1370e-mail: freilich@nso2.uchc.edu
Key words: biomaterial scaffold, ng/rhBMP-2, scaffold retainer, vertical supracrestal bone growth
Abstract
Objective: To attain and describe guided vertical bone regeneration around titanium (Ti) and titanium
zirconium (Ti–Zr) dental implants utilizing non-glycosylated recombinant human bone morphogenetic
protein-2 (ng/rhBMP-2), biomaterial scaffolds and a scaffold retainer.
Materials and methods: Thirty-two modified Straumann TE implants were partially embedded in the
mandibles of eight adult mini-pigs. Pre-shaped resorbable scaffolds were placed around the implant and
shielded and stabilized with a newly developed Ti custom scaffold retainer (umbrella) or wide-neck (WN)
healing caps to stabilize the scaffold. Ng/rhBMP-2 (50mg) was applied to the supracrestal portion of the
implant or incorporated within the scaffold. At 9 weeks, soft tissue healing was assessed. Vertical bone
regeneration outcomes including bone height, bone-to-implant contact (BIC) and bone volume were
assessed by micro-computed tomography and histology.
Results: Soft tissue healing at the test sites (þng/rhBMP-2/þ scaffold) appeared to be substantially
better than the control sites (�ng/rhBMP-2/� scaffold). Bone height, BIC percentage and bone volume
were all similar regardless of whether WN healing caps or umbrella scaffold stabilization was used for all
biomaterial scaffolds tested. WN healing cap test sites showed greater new bone height and BIC as
compared with aggregate data from the control sites (P¼ 0.05). Comparison of aggregate data from the
umbrella test sites showed greater BIC and new bone volume as compared with aggregate data from the
control sites(P¼ 0.05).
Conclusion: Vertical bone regeneration was successfully attained utilizing ng/rhBMP-2, biomaterial
scaffolds and a scaffold retainer.
Current clinical methods for increasing vertical
bone height include grafting from intra- and
extra-oral sites and distraction osteogenesis. A
successful outcome is site dependent and gener-
ally associated with substantial levels of morbid-
ity (Feichtinger et al. 2007). The overall goal of
the study described here is to use dental implants,
implant components and osteoinductive factors
to guide a new layer of bone height while mini-
mizing the technique sensitivity, risks and pro-
blems seen with current clinical methods. The
development of methods to predictably regener-
ate 2–3 mm of additional bone height of good
density and width would have important clinical
implications at both anterior and posterior sites.
The simultaneous placement of a partially in-
serted dental implant, an inductive agent and a
resorbable three-dimensional scaffold has been
attempted to treat the vertical defect in experimen-
tal dog/large animal models with varying degrees
of success (Jovanovic et al. 1995; Renvert et al.
1996; Sigurdsson et al. 1997; Roos-Jansaker et al.
2002). For this approach, inductive agents have
included allograft materials (Jung et al. 2008) or
growth factors such as bone morphogenetic pro-
tein-2 (BMP-2).
A demineralized bone matrix (DBM) allograft
has osteoinductive qualities and can serve as the
three-dimensional scaffold to support new tissue
growth. DBM is able to maintain space for bone
growth, exhibit a framework for cell and matrix
protein adhesion, and has the potential to deliver
osteogenic cell signaling agents. The osteoinduc-
tive value of DBM was recognized almost four
decades ago after its placement into muscle
pouches of animals and the subsequent formation
of ectopic bone (Urist 1965; Lindholm et al. 1988).
It has been shown that the osteogenic components
Date:Accepted 14 March 2011
To cite this article:Freilich M, Wen B, Shafer D, Schleier P, Dard M, Pendrys D,Ortiz D, Kuhn L. Implant guided vertical bone growth in themini-pig.Clin. Oral Impl. Res. xx, 2011; 000–000doi: 10.1111/j.1600-0501.2011.02199.x
c� 2011 John Wiley & Sons A/S 1
of DBM are a mixture of growth factors including
many from the transforming growth factor-b fa-
mily (Urist & Strates 1971) such as BMP-2.
Recombinant human (rh)BMP-2 is a growth
factor that has been used frequently to induce
bone growth in a variety of experimental animal
models (Wikesjo et al. 2008). Furthermore, rhBMP-
2 is now FDA approved and commercially available
for use in the jaws of humans. Most previously
reported work utilized a variety of dosages of the
glycosylated formulation of this agent. For our
studies, we chose to use the non-glycosylated
rhBMP-2 (ng/rhBMP-2) version of this molecule,
which has reduced solubility and inherent delayed
release relative to glycosylated rhBMP-2 (Sampath
et al. 1990; Schmoekel et al. 2004; Sachse et al.
2005). Ng/rhBMP-2 shows promise in terms of
allowing a reduction of BMP-2 doses necessary for
the efficient induction of bone (Woo et al. 2001),
which is important, given the possible safety issues
associated with BMP-2 (Poynton & Lane 2002).
A non-resorbable titanium (Ti) implant surface
can be used to carry an osteogenic agent such as
BMP-2 to enhance local bone formation and os-
teointegration (Hall et al. 2007; Leknes et al. 2008;
Wikesjo et al. 2008). Ng/rhBMP-2 has been re-
leased from miniaturized Ti implant components
to grow a robust new layer of bone in mouse
calvaria (Freilich et al. 2008). Synthetic resorbable
biomaterial scaffolds may also be used effectively
to release an agent to guide bone growth. Healos, a
collagen/hydroxyapatite (HA) composite scaffold
biomaterial (Jþ J, DePuy, Rayhnam, MA, USA),
is composed of cross-linked bovine Type I collagen
fibers coated with approximately 30% HA exhibit-
ing pore sizes of 4–200mm (Jahng et al. 2004).
Outcomes resembling the use of autogenous bone
grafts have been described when this scaffold was
used in conjunction with bone marrow aspirate or
recombinant human growth/differentiation factor-
5 (GDF-5) (Lutolf et al. 2003; Jahng et al. 2004;
Miranda et al. 2005; Neen et al. 2006).
Dental implant surface roughness and positive
charge (SLActivet, Institut Straumann, Basel,
Switzerland) are associated with osteopromotive or
osteoconductive function (Cochran et al. 1996).
These characteristics have been associated with
enhanced osteoblast function and matrix production
as well as increased levels of endogenous growth
factors in cell culture studies (Kieswetter et al. 1996)
and are well suited for guiding new bone growth.
Implants with these surface characteristics may be
machined from commercially pure Ti and more
recently a Titania–zirconia (Ti–Zr) alloy. Enhanced
design features used to stabilize the adjacent resorb-
able scaffold may help to guide vertical osteogenesis.
As a follow-up to earlier mouse studies, a newly
developed custom scaffold retainer (umbrella) was
used to successfully grow a new layer of bone in
rabbit mandibles (Freilich et al. 2009).
The present descriptive study builds upon
previous small-animal research by testing a vari-
ety of inductive agents and scaffolds in an in-
traoral large-animal model. The purpose was to
generate outcomes that would then influence the
therapy selection and inform sample size/power
calculation for subsequent studies. This study
explored the effect of two primary variables on
new bone growth in the mini-pig: (1) the type of
scaffold retainer used (wide–neck [WN] healing
abutment vs. a custom ‘‘umbrella’’ scaffold re-
tainer) and (2) the release of ng/rhBMP-2 from
pre-made fibrous scaffolds (DBM or Healos) or
dental implant surfaces.
Materials and methods
Study design
The study timeline can be seen in Fig. 1. This
study protocol was approved by the ethics com-
mittee for animal research at Lund University in
Sweden. Four implant sites were used in each of
eight adult Gottingen mini-pigs ranging in weight
from 30 to 50kg and 2 years of age. The study
design includes four study groups: one negative
control (two animals and eight implant sites) and
three treatment groups each with two animals and
four implant sites for a total of 32 study sites. Each
treatment group animal (groups 2, 3 and 4) pro-
vided a split-mouth comparison between oversized
WN healing caps and newly developed custom Ti
scaffold retainer (umbrella) abutments. The speci-
fic treatment groups can be seen in Table 1 and
they include: (1) Ti-negative controls without
BMP or scaffold; (2) Ti/BMPþDBM; (3) Ti–Zr/
BMPþHealos; and (4) Ti–ZrþHealos/BMP. The
effects of two primary independent variables were
evaluated. These were: (1) the use of an umbrella
vs. a WN healing cap to stabilize the biomaterial
scaffold and (2) the site of ng/rhBMP-2 release.
Secondarily, the use of Ti vs. a Ti–Zr alloy and
scaffold type was also evaluated. For all sites, the
dental implants were partially embedded in poster-
ior mandible sites such that the implant shoulder
was located 2.5 mm above the bone crest. The
Fig. 1. Time schedule schematic.
Table 1. Chronology of study procedures
Groupn Side A Side B
1 (n¼2) Ti SLA with Ti SLActive with a Controla WN healing cap WN healing cap
2 (n¼2) Ti implant/BMP (50 mg) Ti implant/BMP (50mg) TestDBM scaffold DBM scaffoldUmbrella WN healing cap
3 (n¼2) Ti–Zr implant/BMP (50 mg) Ti–Zr implant/BMP (50mg)Healos scaffold Healos scaffoldUmbrella WN healing cap
4 (n¼2) Ti–Zr implant Ti–Zr implantHealos scaffold/BMP (50 mg) Healos scaffold/BMP (50 mg)Umbrella WN healing cap
nTwo animals per group, two implants per side per animal.
Ti, titanium; BMP, non-glycosylated rhBMP-2; DBM: demineralized bone matrix; Ti–Zr, titanium zirconium; WN,
wide-neck; BMP, bone morphogenetic protein.
Freilich et al � Implant-guided bone growth in mini-pig
2 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02199.x c� 2011 John Wiley & Sons A/S
system was designed to guide the regeneration of
new bone along the aspect of the implant surface
left outside of bone at the time of placement. Ng/
rhBMP-2 was used as an osteoinductive signaling
agent, which was released from either the osteo-
genic portion of the implant surface or the resorb-
able scaffold adjacent to the implant at the
supracrestal region. A titanium umbrella abutment
or a WN healing cap stabilized the scaffold and
provided protected space for new bone formation.
Implant design and manufacturing
The 9 mm long � 4.1 mm solid screw implants
made of commercially pure titanium or a tita-
nium–zirconium (Ti–Zr) alloy (Roxolidt) were
obtained from Institut Straumann. The implants
were cylindrically shaped, except for the coronal-
most 2.5 mm of the implant, which exhibited a
slight taper and a diameter of 4.8 mm at the head of
the implant. A roughened, sand-blasted, large grit,
acid-etched surface (SLAt) or a hydrophilic SLA
surface with a positive charge (SLActivet) was
placed to the finish line at the head of each
implant. There was no polished collar.
Custom-designed titanium scaffold retainer
abutments (umbrella) 9 � 9 mm in size, a thick-
ness of 1 mm with polished surfaces and four
‘‘L’’-shaped openings, and oversized WNHCs
with a diameter of 7 mm and a thickness of
2 mm were also obtained from Institut Strau-
mann. The design of the implant and umbrella
can also be seen in Fig. 1.
Experimental scaffolds
DBM allograft material using procedures similar
to those for producing a commercially available
human allograft material (Graftons
Flex) (Osteo-
tech, Eatontown, NJ, USA) and HA-coated col-
lagen (Healoss
) (DePuy Spine, Raynham, MA,
USA) were both prepared up to a size of
10 � 10 mm, thickness of 3 mm and a central
opening with a diameter of 5 mm, which allowed
for the scaffolds to be placed around the implants.
BMP-2 delivery
Escherichia coli expressed ng/rhBMP-2 was used
as an osteogenic signaling agent in these studies
as described previously in Sachse et al. (2005).
The three different ng/rhBMP-2 delivery systems
tested included a resorbable scaffold (adjacent to
the SLActive surface Zr–Ti implant) and two
different implant surfaces adjacent to non-BMP
carrying scaffolds.
For BMP-2 released from Ti and Zr–Ti SLActive
implant surface (Ti/BMP), lyophilized ng/rhBMP-2
powder was reconstituted in 50% acetonitrile/
0.1% trifluoroacetic acid (ATFA) at a concentra-
tion of 5 mg/ml and placed aseptically onto the
osteogenic aspect (coronal most 2.5 mm) of an
SLActive-treated implant by evenly wetting the
surface of each implant with 10ml of the ng/
rhBMP-2 solution. All samples were dried under
a laminar air flow in a biological safety cabinet.
Each Ti SLActive implant (100mg/cm2 surface)
had an ng/rhBMP-2 dosage of 50mg. As the volume
of the Healos is ten times larger than that of Ti or
the Zr–Ti implant surface, a stock ng/rhBMP-2/
ATFA Solution (5 mg/ml) was diluted to a con-
centration of 1 mg/ml and 50ml of this solution
was applied to the Healos with a Hamilton syringe
(Hamilton, Reno, NV, USA) to obtain the
same dose as that on the Ti or the Zr–Ti implant.
Each scaffold (200 ng/mm3 scaffold) had an ng/
rhBMP-2 dosage of 50mg. All samples were dried
under a laminar airflow in a biological safety
cabinet.
Surgical procedure
The animals were pre-medicated with an intra-
muscular injection of atropine (atropinum sulfur-
icum, 0.05 mg/kg IM) and then given a
combination of ketamine (10 mg/kg, Ketalar
Vet 50 mg/ml, Pfizer AB, Sollentuna, Sweden)
and midazolam (0.5 mg/kg Dormicums
5 mg/
ml: Roche, Basel, Switzerland) for general an-
esthesia. During the surgery, ketamine (10 mg/
kg) and midazolam (0.5 mg/kg) were reinjected
when needed. All animals received 1.8 ml local
anesthesia (Xylocain Dental adrenalin 20 mg/
mlþ12.5 mg/ml, Astra AB, Sodertalje, Sweden)
at each surgical site just before surgery.
The surgical procedures can be seen in Fig. 2
and were performed 6 months after the extraction
of the second, third and fourth pre-molars and the
Fig. 2. Clinical photographs of the surgical procedure. (a and b) Two implants placed in each side of the posterior mandible after
a gentle reduction of the alveolar crest to create a flat surface. Implants placed with the shoulder located 2.5 mm above bone level.
(c, d and e) Scaffolds placed around the supracrestal part of the implant and covered by a umbrella or a wide-neck healing abutment.
Freilich et al � Implant-guided bone growth in mini-pig
c� 2011 John Wiley & Sons A/S 3 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02199.x
first molar. Incisions were made with a 15-size
blade on the edentulous alveolar crest to allow
full-thickness flap elevation. Osteotomy and
implant placement procedures followed the sur-
gical protocol described elsewhere (Dard et al.
2008). The custom-designed 9 � 4.1 mm im-
plants were placed bilaterally to a depth of
6.5 mm, leaving 2.5 mm of each implant beyond
the level of bone. Partial bone decortication
surrounding the implant was made at four evenly
placed sites to enhance blood clot development
and access to intraosseous vasculature and cells.
For test sites with scaffolds, the scaffold was
placed in direct contact with the underlying
partially decorticated bone and maintained in
position by a custom-designed scaffold retention
screw or a WN healing cap fastened to the screw
channel at the head of each implant. Once all
elements of the implant system construct were
placed, the mucoperiosteal flap was closed with
4.0 suture material (Vicryl, Ethicon Inc. Johnson
& Johnson company, Somerville, NJ, USA).
A veterinary technician monitored all vital signs
of each animal during surgery. Postsurgery, all
animals were placed on a soft diet.
Terminal procedures and soft tissue evaluation
All animals were sacrificed 9 weeks after dental
implant placement by inducing a cardiac arrest
with an intracardiac injection of a 20% solution
of pentobarbital (Pentobarbitalnatrium, Apoteket
AB; Stockholm, Sweden, 60 mg/ml) after general
anesthesia. Soft tissue healing was quantified
with scores of 0 (no dehiscence) to 4 (complete
dehiscence) as can be seen in Fig. 3. The mand-
ibles were excised and the left and right hemi-
mandibles were separated with a band saw, fixed
(4% formaldehyde) for 2 weeks and prepared for
micro-computed (micro-CT) tomography ima-
ging and histological processing by transfer into
an ethanol solution.
Micro-CT evaluation
Following fixation, all samples were imaged
three-dimensionally using conebeam X-ray mi-
cro-CT (CT80, Scanco Medical AG, Bassersdorf,
Switzerland). A trilinear interpolation method
was implemented to rotate the micro-CT images.
This resulted in alignment of the implant with
the z-axis in voxel space and acted as a smoothing
filter. Serial tomographic images were acquired
transverse to the implant longitudinal axis at
90 kVp and 77mA, collecting 1000 projec-
tions per rotation at 400 ms integration time.
Three-dimensional cone beam images were re-
constructed with 50 mm nominal resolution (iso-
tropic). The images were segmented to separate
the implant and bone from the background using
a global thresholding procedure. Newly regener-
ated supracrestal bone height and volume were
measured directly from the segmented images.
Histopathologic and histomorphometric analyses
To identify the position of the sites, X-rays were
performed before histological preparation. The
samples were dehydrated in alcohol of increasing
concentrations, cleared in xylene and embedded
in polymethylmetacrylate resin. For each hemi-
mandible, one sagittal mesio-distal and two
bucco-lingual histological sections were prepared.
The sections were obtained using a micro-cutting
and grinding technique adapted from Donath
(Donath & Breuner 1982). The sections were
then stained for qualitative and quantitative his-
Fig. 3. Soft tissue summary and scores (groups 2, 3 and 4 – Umbrella vs. wide-neck healing cap [WNHC]).
Fig. 4. Representative histology images (groups 2, 3 and 4 – Umbrella vs. wide-neck healing cap [WNHC]).
Freilich et al � Implant-guided bone growth in mini-pig
4 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02199.x c� 2011 John Wiley & Sons A/S
tology with a modified polychromatic Paragon
staining. The histological sections were observed
using a Nikon microscope (Eclipse E600, Nikon,
Melville, NY, USA) fitted with � 2, � 4, � 10,
� 20 and � 40 lenses. Photomicrographs were
performed. The stained ground sections were
observed using a Zeiss Axioscope microscope
(Thornwood, NY, USA) fitted with � 5, � 10,
� 20 and � 40 objectives and equipped with a
color image-analyzing system SAMBAs
(SAMBA
Technologies, Chatillon, France). The outcomes
adjacent to each implant were assessed by analyz-
ing bone growth, osseointegration and the
presence or absence of neovascularization. Quali-
tative and semi-quantitative analyses were per-
formed to measure the height of supracrestal bone
at the implant surface (mm), which was measured
from just external to the cortical surface of the
underlying bone to the highest point of bone-to-
implant contact (BIC) % to new bone. Addition-
ally, the BIC was determined at both the osteo-
genic (supracrestal bone) and the anchoring
(subcrestal) portions of the implant within one
mm of the implant surface.
Data analyses
Data analyses were performed using SPSS soft-
ware, version 18 (IBM Corporation, Armonk,
NY, USA). Descriptive statistics, including
means, standard deviations and distribution
shape, were generated for all outcome measures.
The unit for all analyses was the mini-pig. As
each mini-pig had two implants per side, the
average values for each implant pair were used
for all analyses. Between-group analyses were
conducted for both micro-CT and histology out-
comes using the Mann–Whitney U-test for in-
dependent samples.
Results
Uneventful healing and recovery of all animals
followed the surgical procedures. All outcomes
were assessed at 9 weeks postimplant placement.
All of the negative control sites had a soft tissue
dehisence whereas only four of the 24 of the
implants with BMP and scaffold (groups 2, 3, 4)
sites had dehisence. The semi-quantitative
findings and a summary of the scores for soft tissue
dehiscence comparing umbrella and WN healing
caps can be seen in Fig. 3. There did not appear to
be any meaningful difference between umbrella and
WN healing cap sites for any of the treatment pairs.
Qualitiative histology showed that the newly
formed bone tissue was characterized by woven
bone covered by a thin layer of cortical bone. The
newly formed bone merged with the original
alveolar bone, with substantial vasculature infil-
trating the marrow and bone tissues. At the
cellular level, osteoblasts actively depositing
bone were observed throughout the newly formed
bone as were few inflammatory cells. Represen-
tative histology sections of ‘‘test’’ treatment
groups 2, 3 and 4 all showing substantial vertical
bone regeneration can be seen in Fig. 4.
Bone height
Fig. 5a shows new bone height data as deter-
mined via quantitative histology. The findings
for negative control group sides 1A and 1B were
similar and thus combined. Umbrella and WN
healing cap findings within the test sites (groups
2, 3 and 4) were also generally similar. A com-
parison of aggregate data for mean new bone
height (mean¼0.62 mm, SD¼0.07) from the
negative control sites (n¼2) with aggregate data
from BMP/scaffold test sites in groups 2, 3 and
4 that used a WN healing cap (n¼6) demon-
strated more than a three-fold increase for the test
sites (mean¼1.97 mm, SD¼ .74, P¼0.04).
New mean bone height for umbrella test sites
in groups 2, 3 and 4 (n¼6) was 1.75 mm
(SD¼0.28), slightly less than a threefold increase
as compared with the control sites (P¼0.05).
BIC percentage
Fig. 5b shows BIC data as determined via histol-
ogy. As with bone height, the BIC outcomes for
negative control group sides 1A and 1B were
similar and thus combined. All active treatment
sites (groups 2, 3 and 4) showed similar BIC
values. New mean BIC for aggregate data from
the WN healing cap test sites for groups 2, 3 and
4 (mean¼49.33, SD¼9.22) was 62% greater
than that for the aggregate data of the control
(group 1A and 1B) sites (mean¼30.5%, SD¼0.71)
(P¼0.04). New mean BIC for the group 2, 3 and 4
umbrella test sites (mean¼47.83%, SD¼8.18)
was 57% greater than the negative controls
(P¼0.04).
Bone volume
Fig. 5c shows new mean bone volume data as
determined via micro-CT. As with bone height
and BIC, bone volumes for negative control group
sides 1A and 1B were similar and were thus
combined. Umbrella and WN healing cap find-
ings within the test treatment sites (groups 2, 3
and 4) showed similar new mean bone volume.
New mean bone volume for aggregate data from
the WN healing cap test sites for groups 2, 3 and
4 (mean¼106.41 mm3, SD¼22.39) was 65%
greater than that of the aggregate data of the
control groups (mean¼64.31, SD¼14.70)
(P¼0.1). New mean bone volume for the group
2, 3 and 4 test sites with umbrella was 59%
greater (mean¼102.39 mm3, SD¼13.24) than
that seen with the negative controls (P¼0.05).
Representative micro-CT images for the control
and the active treatment groups, 2, 3 and 4 can be
seen in Figs 6 and 7, respectively.
Discussion
In this descriptive study, implant-guided bone
regeneration in association with an osteogenic
Fig. 5. Histology and micro-CT Outcomes, (a) bone height as measured by histology, (b) bone-to-implant contact percent as
measured by histology, (c) bone volume as determined by micro-CT.
Freilich et al � Implant-guided bone growth in mini-pig
c� 2011 John Wiley & Sons A/S 5 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02199.x
agent, ng/rhBMP-2, a biomaterial scaffold and a
scaffold retention device was evaluated for the
first time in a large-animal intraoral mini-pig
model. Previously, successful results were ob-
tained when testing this dental implant bone
regeneration system in several small-animal
models (e.g. mouse, rat and rabbit models [Frei-
lich et al. 2008, 2009]); however, those models
were not intraoral models. The present study
shows that the release of ng/rhBMP-2 from the
dental implant surface or scaffold materials in
combination with the use of a scaffold-stabilizing
device reliably induced approximately 2 mm of
vertical bone growth of good density and width
along the length of the dental implant. Many
clinical situations requiring implant placement
would best be managed by the addition of new
alveolar bone height and volume. Based on these
studies, the use of a custom scaffold retainer
(umbrella) or a WN healing abutment to stabilize
and retain pre-made fibrous scaffolds (DBM and
Healos) in the presence of ng/rhBMP-2 around Ti
and Ti–Zr implants appears to be a suitable
option to increase alveolar bone height. In this
study, histology and micro-CT analysis deter-
mined that the negative control sites (�BMP
� scaffold) utilizing WN healing abutments and
implants without a scaffold and ng/rhBMP-2
regenerated very little new bone. In contrast,
the test groups with ng/rhBMP-2 and any
of the resorbable scaffold biomaterials (þBMP
þ scaffold) were able to effectively support a new
layer of wide, dense vertical bone growth.
The findings of this study suggest that supra
alveolar bone can be grown with a high level of
predictability as demonstrated by new bone aug-
mentation across all of our umbrella and WN
healing cap test groups. Quantitative histology
confirmed that new supracrestal bone growth for
all þBMPþ scaffold groups was of good quality
and density and was well adapted to the Ti and
Ti–Zr implant surfaces. Micro-CT showed that
considerable new bone volume regenerated at all
of these test sites. The data further show that the
type of scaffold stabilizing device (WN healing
cap vs. umbrella) and site of ng/rhBMP-2 release
(implant surface vs. scaffold) had minimal prac-
tical influence on bone height, BIC percentage or
bone volume at the test sites. A striking differ-
ence was also seen in soft tissue healing for the
experimental sites in contrast to the control sites.
All of the control sites, which had neither BMP nor
a scaffold (group 1A and 1B), exhibited soft tissue
dehisence, whereas only four of the 24 sites with
BMP and scaffold (groups 2, 3, 4) had dehisence.
No practical difference was seen in the osteoinduc-
tive efficacy of bone formation between two types
of scaffold holding devices in this study, which
used fibrous, pre-made scaffolds. This might be
explained by the fact that the 6.5mm WN healing
cap also served as a scaffold retention screw to
maintain space and keep scaffold biomaterials
stable, while at the same time providing protection
from soft tissue collapse and traumatic overload-
ing. The additional size and extension of the
umbrella beyond that of the WN healing abutment
would allow it to confer effective retention and
stability to particulate scaffold materials that may
be selected for use in future studies.
Previous studies have made attempts to utilize
dental implants in the absence of autogenous
grafts for the purpose of guiding vertical bone
growth. These studies have used occlusive mem-
branes such as polytetrafluoroethylene (PTFE)
with and without titanium reinforcement to
maintain space and prevent soft tissue cells
from repopulating the area of attempted bone
regeneration. Unsuccessful attempts have in-
volved the use of a blood clot in the absence of
a scaffold or an agent to induce new bone forma-
tion in a dog model (Roos-Jansaker et al. 2002;
Polimeni et al. 2005). Later testing utilizing the
same approach, but with the addition of glycosy-
lated BMP-2, produced a narrow dimension of
vertical bone growth of low density (Wikesjo
et al. 2008). Studies in the dog model (Jovanovic
et al. 1995; Renvert et al. 1996; Sigurdsson et al.
1997) and case reports in humans (Tinti et al.
1997; Simion et al. 2007) have reported better
outcomes for this approach. These previous stu-
dies utilize the technique-sensitive PTFE mem-
brane, which is likely to result in the failure of
new bone regeneration when soft tissue dehis-
cence results in membrane exposure.
The present study utilized two osteoinductive
carrier systems: (1) a rough Ti surface and (2)
Healos. It has been demonstrated previously that
21mg of ng/rhBMP-2 released from the roughened
(SLA) surface of a non-resorbable Ti scaffold can
induce substantial bone formation in a murine
calvarial model (Freilich et al. 2008). The positive
outcomes with 50mg of the ng/rhBMP-2 released
from rough Ti surfaces seen in the current study
are consistent with the outcomes achieved in an
earlier canine intraoral study that demonstrated
alveolar ridge augmentation using 200 or 600mg
of glycosylated rhBMP-2 coated on the titanium
porous-oxide surface (Leknes et al. 2008; Wikesjo
et al. 2008). The use of ng/rhBMP-2 allowed
positive results to be achieved using a substan-
tially lower dosage. This is confirmed in work by
others utilizing release from a fibrin matrix
(Schmoekel et al. 2005). Healos has been suc-
cessfully used in previous work as a scaffold in
Fig. 7. Representative microcomputed tomography images (groups 2, 3 and 4 – Umbrella vs. wide-neck Healing cap
[WNHC]). The green line represents the approximate original bone level.
Fig. 6. Representative microcomputed tomography images (group 1). WNHC, wide-neck healing cap.
Freilich et al � Implant-guided bone growth in mini-pig
6 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02199.x c� 2011 John Wiley & Sons A/S
conjunction with the infusion of bone marrow
aspirate or by releasing recombinant human
GDF-5 (Jahng et al. 2004; Magit et al. 2006).
However, this study is the first to demonstrate
that Healos can successfully carry ng/rhBMP-2.
Although the sample size for the present
study was small and power was low, it was
possible to demonstrate a new layer of vertical
bone growth, with clear differences seen between
the test and the control groups. The findings
obtained by this descriptive study help to estab-
lish the foundation for future studies with a larger
sample size and fewer variables in the same
animal model.
Acknowledgments: This study was
supported by Institut Straumann AG, Basel,
Switzerland, which included the manufacture
of custom experimental titanium implants and
components. We appreciate the gift of ng/
rhBMP-2 provided by Dr Peter Hortschansky of
the Hans Knoll Institute, Jena, Germany, and
the excellent micro-CT imaging performed by
Dr Martin Stauber at b-cube AG, Zurich,
Switzerland. We also thank Dr Antoine Alves
(NAMSA) for histological and
histomorphometric analysis. Finally, we are
highly indebted to Mr Marcel Obrecht at
Institut Straumann for his careful organization
of all activities during surgical implantation
and important contributions to this
manuscript.
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c� 2011 John Wiley & Sons A/S 7 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02199.x