Oral Maxillofac Surg (2014) 18:355–372
DOI 10.1007/s10006-013-0397-2
REVIEW ARTICLE
Implants in bone: Part II. Research on implant osseointegration
Material testing, mechanical testing, imaging and histoanalytical methods
Cornelius von Wilmowsky & Tobias Moest &
Emeka Nkenke & Florian Stelzle & Karl Andreas Schlegel
Received: 29 January 2013 / Accepted: 4 February 2013 / Published online: 21 February 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract
Purpose In order to determine whether a newly developed
implant material conforms to the requirements of biocom-
patibility, it must undergo rigorous testing. To correctly
interpret the results of studies on implant material osseoin-
tegration, it is necessary to have a sound understanding of
all the testing methods. The aim of this overview is to
elucidate the methods that are used for the experimental
evaluation of the osseointegration of implant materials.
Discussion In recent decades, there has been a constant
proliferation of new materials and surface modifications in
the field of dental implants. This continuous development of
innovative biomaterials requires a precise and detailed eval-
uation in terms of biocompatibility and implant healing
before clinical use. The current gold standard is in vivo
animal testing on well validated animal models. However,
long-term outcome studies on patients have to follow to
finally validate and show patient benefit.
Conclusion No experimental set-up can provide answers for
all possible research questions. However, a certain transfer-
ability of the results to humans might be possible if the
experimental set-up is carefully chosen for the aspects and
questions being investigated. To enhance the implant sur-
vival rate in the rising number of patients with chronic
diseases which compromise wound healing and osseointe-
gration, dental implant research on compromised animal
models will further gain importance in future.
Keywords Implants . Biocompability . Osseointegration .
Bone . In vivo
C. von Wilmowsky (*) : T. Moest : E. Nkenke : F. Stelzle :
K. A. Schlegel
Mund-,Kiefer- und Gesichtschirurgische Klinik
Universitätsklinikum Erlangen, Glückstrasse 11,
91054 Erlangen, Germany
e-mail: cornelius.vonwilmowsky@uk-erlangen.de
Material testing
In order to determine whether a newly developed implant
material conforms to the requirements of biocompatability,
mechanical stability, and saftey, it must undergo rigorous
testing, both in vitro and in vivo. The evaluation of implant
materials is done by application and evaluation in an animal
model, and then clinical trials in humans.
The hemostasis of the immune response and repair func-
tions in the body are complex, and it is not adequate to
describe the biocompability of a single material in relation
to a single cell type or tissue. In vitro tests are mostly used in
accordance with ISO 10993 to determine the biocompatibil-
ity of a certain material.
ISO 10993 is a standard series for the biological evaluation
of medical products. The aim is the evaluation of the biocom-
patibility of materials implanted in the human body. Thus, not
only medical implants are tested but also basic materials used
for the production of medical products. Along with biological
evaluation, this standard series additionally contains the phys-
icochemical examinations and analyses of materials and sub-
stances under examination and prescribes the maximum
permissible value of bio-active substances.
These tests do not determine the biocompability of a ma-
terial, but they constitute an important step forward to animal
testing and, finally, clinical trials that will determine the bio-
compatibility of the material in a given application [1]. Be-
cause the results of in vitro studies do not reflect the
complexity of the in vivo situation, animal studies remain
necessary. The use of animal models is normally an essential
step in testing dental implants prior to clinical use in humans.
In vivo testing
Animal experiments have always been a proven method for
scientists to evaluate and examine hypotheses and clinical
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findings in biomedical research [2]. Most findings in general
biochemistry, physiology, endocrinology and pharmacy as
well as advances in surgical techniques, originate from
animal experiments [3]. Animals are used in biomedical
research as living models to obtain results that cannot be
gained for various reasons in humans. The importance of
these findings for the model, and thus for humans, is depen-
dent on the standardization, reproducibility, and evaluation
of the experimental results [4]. There must be a close anal-
ogy between the animal model and the human organism.
The animal can only be an experimental model, and trans-
ferability to the human organism is only possible with great
care [3, 5]. In biomedical research, animal models are es-
sential for understanding biological pathways and regula-
tion. The experimental animal species used, often differ
qualitatively and quantitatively in regards to morphology,
physiology, or pathology. Nevertheless, a certain transfer-
ability of the results to humans might be possible if the
animal model is chosen carefully for the aspects and ques-
tions being investigated [4]. Confirming the experimental
findings in humans is then the task of clinical research.
However, the findings and results of accuratly planned
animal experiments for all biological questions is not repla-
cable by any other method, such as isolated organs or cell
culture.
Animal models
The use of animal testing is essential for the development of
new implants in dentistry. The choice of the animal speci-
men is a crucial factor for evaluating the biocompatibility
and integration properties of implants and bone substitution
materials (BSM) [6]. The selection of an appropriate exper-
imental animal model analogous to human biology and bone
repair is a prerequisite for the transferability of the experi-
mental results to clinical applications [7].
The rabbit is a suitable animal model for basic research
[8]. An often used and standardized in vivo system to
investigate osseointegration of dental implants, is the rabbit
patellar groove model (Fig. 1) [9–11].
The suitability of this model has been shown in several
industrial studies of bone implants, and it is accepted by the
US Food and Drug Administration for the approval of new
bone implant materials [12]. The advantage of this animal
model is the ease of handling due to its size. Furthermore,
the rabbit reaches skeletal maturity shortly after sexual
maturity at around 6 months of age [13]. Skeletal maturity
represents a fundamental requirement to characterise bone
regeneration or osseointegration of dental implants reliably.
Nevertheless, minimal differences between human and
rabbits bone composition and density exist. A drawback of
this animal model in the assessment of multiple implant
materials is its small size, and the size differences between
Oral Maxillofac Surg (2014) 18:355–372
the bony anatomy of humans and rabbits are essential. The
long bones of the rabbit have a different secondary bone
structure compared to humans [14].
However, the faster skeletal changes and bone turnover
of rabbits in comparison to humans makes it difficult to
draw conclusions for humans [14].
Similar to the rabbit, rats and mice are also used for
biomaterial research in implant dentistry. Due to the size
of the rodents, they have similar advantages and disadvan-
tages as rabbits, but a major drawback remains the very
brisk bone healing and the plexiform bone of these animal
models [6, 8, 15].
Rodents, which were used for research in implant den-
tistry, were mainly used as extraoral models to investigate
healing and soft tissue integration of implants [16].
The pig seems to be the best suited animal, especially for
investigating vascular disease, bone formation, and the bio-
logical behavior of BSM and dental implants, due to its
advantages in comparative experimental medicine [17].
The pig is said to be a valuable model in biomedical re-
search because of its anatomical, physiological, and meta-
bolic similarities to humans [18–21]. The pig has become a
popular animal model to investigate the mechanisms under-
lying bone formation [22]. Its rate of bone regeneration
(1.2–1.5 mm/day) is comparable to that of humans (1.0–
1.5 mm/day), and it is better suited for human transferability
of results compared to all other animal models [18, 23]. The
domestic pig has already proven its suitability for testing
medical implants, BSM, and conventional and biologically
active implant coatings [22, 24] (Figs. 2 and 3). The pig
represents an appropriate model to examine healing and
tissue integration of dental implants in the healthy and the
compromised animal model. To investigate bone healing,
consolidation of bone grafts and the progresses of osseoin-
tegration of dental implants, the pig's forehead represents
the preferred site [25–27]. For surgeons, the preparation of
the forehead is easy to perform and the pig's forehead offers
enough space to prepare several defects or to insert several
implants.
Preclinical studies compared bone regeneration in a por-
cine experimental model with the clinical outcome in
humans after the application of autogenous bone and two
different bone substitutes [28]. It was hypothesized that
there is no significant difference between the bone regener-
ation after application in monocortical critical size defects
on the pig’s forehead and for sinus elevation in humans. The
intention was to demonstrate that the porcine calvarian
defect model simulates bone regeneration in human jaws.
The results of studies about the critical size defect showed
that no significant microradiographic differences in the
quantity of newly formed bone after the application of
autogenous bone or BSM, or in the degree of material
degradation in the animal specimens compared to the human
Oral Maxillofac Surg (2014) 18:355–372
Fig. 1 The rabbit patellar
groove model is often used
as a standardized in vivo
system to evaluate implant
osseointegration. Right: X-ray
photo of the joint with an
inserted metal implant
bone specimens exists. These results clearly demonstrate
that the morphological and anatomical characteristics of
porcine bone produce results that are comparable to humans.
In conclusion, the data demonstrate that the porcine model is
a suitable model for evaluating BSM before clinical use in
maxillofacial and orthopedic surgery. For investigations in
the field of implant dentistry also intra oral models were
applied. For the treatment of mucositis and peri-implantitis
the pig model is not commonly used [16].
The dog is one of the large animal species more frequent-
ly used for musculoskeletal and implant research. One of the
favored dog species for in vivo investigations of implants
and BSM are beagles [29]. The bone exhibits a secondary
osteon structure that can also be found in humans [21].
Furthermore, human and dog cortical and cancellous bone
are similar in terms of the water, organic, volatile inorganic,
and ash fractions [30]. In terms of bone density, the dog and
pig most closely represent humans [23]. Although there are
structural similarities between dogs and humans, however,
in regards to trabecular bone turnover, it is difficult to make
an exact comparison of bone turnover between these species
from the literature reports [31]. Studies show that the vari-
ability of trabecular bone turnover is high and depends on
the bone site [31]. The lumbar vertebral body has a bone
turnover rate of close to 200 % per year in young adult male
beagles, whereas the turnover of the talus is 12 % per year.
Not only is trabecular bone turnover variable between bone
sites within the one individual, there is also large variation in
trabecular bone turnover between individuals, for example
Fig. 2 A monocortical critical
size defect (CSD) in the frontal
skull of the domestic pig to test
bone substitution materials
(BSM) (a). b The location of
the CSD in the frontal skull of
the pig (c). The right picture
shows an X-ray of the BSM in
the CSD after 7 days
357
the mean turnover rate of bone taken by transilial biopsy
from young adult female beagles varies from 16 % per year
to over 300 % per year [31]. Remodelling of the total bone
mass per year for humans is given as 5–15 %, with estimates
of the whole body trabecular bone turnover rate ranging
from 10 % to 15 % per year to 40–55 % [31]. A literature
overview shows that the dog is a good animal model. The
dog model is suitable for intraoral research models to inves-
tigate on healing/tissue integration, healing of compromised
sites and treatment of mucositis/peri-implantitis. For inves-
tigations of tissue healing and integration the dog model is
rarely used [16].
The sheep is an often used animal model and has proven
to be suitable for experimental research on fracture healing
[32]. Primary bone healing and Haversian system remodel-
ing in the sheep has already been described extensively [6].
For research on the long bones of the lower extremities, the
sheep is a useful animal model because the tibia is in the
same axial loading as that in humans [15]. The form and
structure of the tibiae resemble each other, suggesting a
similar load function as described by Wolff's law [15].
Even though the extremities of sheep bear a relatively
close resemblance to human bones, differences have been
found regarding bone structure [33]. Sheep bone is signifi-
cantly more dense and, subsequently, stronger than human
bone [34]. The trabecular bone density was found to be 1.5–
2 times greater than that of humans [35, 36]. On the other
hand, the sheep seems to be a valuable animal model for
osteoporosis research because the bone volume, osteoid
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Fig. 3 a The location of the CSD in the frontal skull of the pig. b Operative site of the CSD after preparation with a threpan drill. c,d The defects
are filled with BSM
volume, and mineral apposition rate of 9- to 10-year-old
ewes are comparable to those of men and post-menopausal
women in their sixth to seventh decade of life [37]. Never-
theless the sheep model represents a common animal model
in the field of dental implant research. Therefore, sheep
were commonly used to investigate healing and tissue inte-
gration of implants in healthy and compromised wound
healing situations, and the extraoral models predominate.
For the treatment of mucositis and peri-implantitis, the
sheep model is not suitable [16].
Summarizing the present knowledge, many different an-
imal models with several advantages and disadvantages
were used in preclinical studies to understand bone regen-
eration and osseointegration of dental implants. Unfortu-
nately, no animal model fulfills the ideal experimental
model, which could be wholly applied (100 %) on humans.
The choice of the respective model depends on the experi-
mental design of the in vivo study and the defined research
question.
Compromised animal models
Dental implants have a predictable and high survival rate
[38–40]. However, clinicians are confronted with an in-
creasing number of medically compromised patients who
require implant surgery for rehabilitation [41]. Diabetes
mellitus, osteoporosis, radiation therapy and bisphosphonate
(BP) medication are common clinical settings, which could
negatively influence bone regeneration capacity and implant
success. For example, more than 150 million people world-
wide have diabetes. In addition, the number of diabetic
patients is constantly rising and the prevalence of diabetes
is expected to double by the year of 2030 [42]. Therefore,
the amount of diabetic patients seeking any medical treat-
ment will grow steadily. Even though there has been inten-
sive research analyzing the success and failure rates of
implants in diabetic patients, only experimental animal stud-
ies allow for a systematic evaluation of the biological and
Oral Maxillofac Surg (2014) 18:355–372
pathological effects of diabetes on implants and medical
device osseointegration [43] (Fig. 12). To understand the
biological, pathological, and molecular mechanisms under-
lying the failure of implants and medical devices in patients
with compromised wound healing, a reliable animal model
would be beneficial. Thus, compromised animal models are
gaining popularity [44–46]; however, these studies deal with
one fundamental problem — the choice of the animal mod-
el. In the case of evaluating the osseointegration of bioma-
terials, the pig seems to be the best suited animal, especially
for investigating vascular diseases, bone formation, and the
biological behavior of implants due to its anatomical, phys-
iological, and metabolic similarities with humans [19, 20,
47]. Furthermore, the domestic pig has already proven its
suitability for testing medical implants, BSM, and conven-
tional and biologically active implant coatings [24, 26, 48,
49]. Because of the good transferability of the results on
humans and the growing demand of a compromised animal
model, the model of a diabetic domestic pig has been
established [47]. With the help of a β-cell toxic drug (e.g.,
Streptozotocin), it is possible to induce an experimental
diabetes leading to significant pathological changes in the
hard and soft tissue. All clinical internal parameters, deter-
mined by the American Diabetes Association, for the defi-
nition of a diabetes mellitus could be fulfilled [47]. Based on
this compromised animal model, the effect of the experi-
mental diabetes on the osseointegration of dental implants
has been investigated [49]. The results show a significantly
reduced bone–implant contact (BIC) and bone mineral den-
sity (BMD) in the diabetic group. It can be concluded,that
poorly controlled diabetes negatively affects peri-implant
bone formation and bone mineralization [49]. However,
the probability of a successful implant osseointegration in
diabetic patients can be improved by using dental implants
with chemical-modificated surfaces [48]. Due to the mod-
ified surface, an accelerated osseointegration with signif-
icantly higher BIC and peri-implant bone density could
be observed. In summary, it can be proposed that the use
Oral Maxillofac Surg (2014) 18:355–372
of dental implants with chemical modified surfaces have
a better prognosis for implant treatment of diabetic
patients [48].
However, there are many more diseases that clinicians
deal with that affect the osseointegration dental implants in
the body; for example, osteoporosis. Ovariectomy has been
used as an experimental model for studying the pathogene-
sis of postmenopausal osteoporosis [50]. After estrogen
withdrawal, the hematopoietic environment in the bone
marrow is altered, leading to the up-regulation of osteoclas-
togenesis. A well recognized animal model for osteoporosis
is the sheep, which has been validated as a large animal
model for the study of vertebral osteoporosis. Reduced
mechanical competence in the lumbar spine confirmed the
suitability of this model for evaluating potential treatments
for osteoporosis [51, 52]. On the other hand, Egermann et al.
[53] reviewed osteoporosis animal models and found that
ovariectomy alone does not cause the bone loss seen in
human patients with the disorder. The magnitude of bone
loss by the attempt to induce osteoporosis by ovariectomy
does not reach the WHO threshold level. To achieve a
substantial bone loss, it is recommended to perfom ovariec-
tomies on geriatric sheeps, additional to a glucocorticoid
treatment and vitamin D/Ca2+ restricted feed [54, 55]. An
other possibility to induce osteoporosis is described by
performing a 7-month glucocorticoid treatment with malnu-
trition. Three months after glucocorticoid cessation, signif-
icant impact on the cortical micro-architecture of the sheep
femur midshaft can be observed [56]. Therefore, studies
with the osteoporotic sheep confirm that in case of osteope-
nia, both bone formation and maturation are delayed. Thus,
a careful evaluation of the healing rate and bone maturation
degree around implanted biomaterials is recommended [57].
Furthermore, numerous pathological conditions, such as
reduced blood micro-circulation after, therapeutic irradiation
therapy, can lead to dental implant failure [58, 59]. There-
fore, several animal models have been established to inves-
tigate the effects of irradiation on bone and dental implant
outcomes. Thereby osseointegration of dental implants is
analysed in irradiated bone of beagle dogs, minipigs and
rabbits [60–64]. Through these models, it was demonstrated
that radiation affects the mineralized bony substrate and,
thereby, the outcome of implant treatment in humans. Also,
the risk of implant failure in irradiated bone is 2–3 times
greater than in non-irradiated bone [59].
This is a serious complication following radiation thera-
py, with or without surgical intervention, for malignancies
of the head and neck is osteoradionecrosis (ORN) of the
mandible [65]. Because the exact pathogenesis of ORN
remains to be elucidated and is a prerequisite for an exact
determination of its pathogenesis, the regenerative potential
and osseointegration of implant materials requires the avail-
ability of an experimental irradiation model. To understand
359
the mechanism underlying jaw ORN and bone regeneration
after radiotherapy animal models in the pig, rat and rabbit
have been established [66–69].
Fenner et al. determined the effect of high-dose irradia-
tion on the rat mandible in order to establish a compromised
experimental animal model of radiogenic bone damage [68]
(Fig. 4).
External irradiation with a total reference dose of 60 Gy
was shown in rats to produce all histological changes attrib-
uted to ORN, after 6 weeks. This irradiation protocol is
suitable for the assessment of regenerative options in severe
radiogenic bone damage [68]. A recent clinical systematic
review showed similar failure rates for dental implants
placed pre- radiography compared with those placed post-
radiotherapy — 3.2 % and 5.4 %, respectively [70]. It was
observed that implant failure rate was significantly higher in
the maxilla compared with the mandible. Furthermore, it
could be demonstrated that implant loss occures within
3 years after radiotherapy but most within 1 to 12 months.
No implant failures were reported when radiation dose was
less than 45 Gy [70]. The survival rate after 8 years was
95 % in non-irradiated residual bone, whereas in irradiated
residual bone 72 % of the implants were osseointegrated.
All implants were placed post-radiotherapy and the patients
received 50 Gy of radiotherapy [71]. Based on the survival
data in literature no specific implant could be recommended
for use in the irradiated upper or lower jaw [72].
As well as radiotherapy, osteonecrosis of the jaw has
been described in patients taking BP after a surgical proce-
dure or dental implant placement. Oral and intravenous BP
medication can be distinguished. Oral BP are used in the
treatment of patients with osteoporosis or other bone dis-
eases like Paget’s disease. Intravenous BP are administered
to patients with breast cancer, multiple myeloma, bone
metastasis to increase survival and quality of life. BP bond
with bone and inhibit osteoclast activity and induce their
apoptosis. The main complication associated with the BP
medication is osteonecrosis of the jaw, which depends on
the strength and the half life of the BP. Experimental studies
in animal models treated with BP observed an improvement
of osseointegration of dental implants unlike the results
obtained in human were osteonecrosis were observed. Rea-
sons for a lack of similarity with humans are thought to be
due to: a short medication and follow-up period after BP
medication, the fact that predominantly the dental implant
were not placed in oral cavity, the only location where BP
associated necrosis are described and not a randomised
controlled trial, was with a large animal model which had
a good transferability to humans, but not yet published.
Each of the presented animal models has advantages and
disadvantages. All of these models have contributed to
further improvements in the diagnosis and treatment of
diseases. The compilation of compromised animal models
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Fig. 4 Identification of target
volume in the jaw of a rat, as
indicated in a, for stereotactic
radiosurgery in b CT scans
performed prior to the first
fraction. Target volume and
100 % isodose are marked pink.
c Mandibular bone of an
irradiated rat (60 Gy) after
12 weeks. The cancellous bone
architecture is irregular with a
higher amount of fibrous tissue
formation (*) compared to d the
healthy control group Staining
is elastica/van Giesson
of different species and with different experimental setups
remains important for investigating the influence of these
factors on results. Yet, there is no animal model which is
ideal.
Evaluation of implant osseointegration
Successful osseointegration, as described in part I is a com-
bination of direct structural and functional connections
existing between ordered, living bone and the surface of a
load-carrying implant [73]. The histological appearance of
the bone–implant interface resembles functional ankylosis
with no intervening fibrous tissue between the bone and
implant [74]. Several tests have been established to evaluate
the degree of osseointegration of a dental implant.
Mechanical tests
Osseointegration is also a measure of implant stability, but
two types of stability have to be differentiated: primary
stability and secondary stability of the implant [75]. The
primary stability of an implant essentially depends on its
mechanical engagement with the cortical and cancellous
bone. Secure cortical stability leads to predictable cancel-
lous stability, which increases after 4 weeks after implant
insertion [76, 77]. At this point, the lowest implant stability
is expected, due to cortical bone resorption after implant
insertion leading to a decreasing primary stability.
Oral Maxillofac Surg (2014) 18:355–372
Secondary stability attains its stability from biological pro-
cesses and depends on peri-implant bone formation and bone
remodeling processes [78]. These progresses can be observed a
few weeks after implant insertion. Bone remodeling is a the
process for secondary stability, whereas secondary implant sta-
bility dictates the functional loading time [79]. This stability also
depends on the quality of the surrounding bone. Therefore, it is
important that implant stability is quantitatively measurable at
different time points. Thus, a long-term prognosis regarding
implant stability is based on stability measurements. Consecu-
tively, different measuring methods for defining primary and
secondary stability of implants have been described. The listed
methods are subdivided in measuring methods specialised on
primary stability and measuring methods which are appropiate
to determine primary and secondary stability as well.
Primary implant stability
Surgeon’s perception One method to evaluate primary sta-
bility is the perception of the surgeon [80]. This is based on
the cutting resistance and seating torque of the implant
during insertion. A perception of “good” stability may be
heightened by the sensation of an abrupt stop when the
implant is seated. The disadvantages of this method are that,
on one hand, surgeon’s perception is invaluable and on the
other hand that the perception is not quantifiable. Most
important, this measuring method can only be made during
implant insertion — it cannot be used before implant load-
ing, for example.
Oral Maxillofac Surg (2014) 18:355–372
Cutting torque resistance analysis The essential condition
of implant insertion is the preparation of a bone socket
appropriate to the implant shape. During the process of
preparation a cutting analysis can be drawn up. During
cutting resistance analysis, the energy (J/mm3), required
for a current fed electric motor to cut off the volume of
bone, is measured. Thereby the energy correlates with the
bone density, and the bone density represents a crucial factor
that considerably affects implant stability [81]. In clinical
use, cutting resistance analysis has been shown to objective-
ly estimate bone density. Therefore, cutting resistance anal-
ysis can be used to measure the primary stability of an
implant. However, it is not possible to make a statement
about the bone quality and evaluate changes in the peri-
implant bone quality after implant placement [82]. Howev-
er, cutting torque resistance analysis cannot identify the
lower "critical" limit of cutting torque value [81].
Insertion torque Measuring insertion torque during installa-
tion of the implant is an attempt to quantify the surgeon’s
tactile perception. The maximum value can be used to
predict the initial BIC; with help of this information the
clinician may provide the optimal loading time [83]. A
disadvantage of this method is that the insertion torque
varies depending on the cutting properties of the implant
and the presence of fluid in the preparation. In this process,
the implant shape has no influence on the insertion torque
[84]. However, the method does yield some information
about the energy used when installing the implant. Higher
energy needed for insertion, represents higher bone density,
which correlates with higher primary stability and the clin-
ical outcome [85]. The main disadvantages are, like the
surgeon’s perception, that insertion torque measurements
can only be used when the implant is inserted and not at
later stage of the treatment process. To obtain a favourable
clinical outcome, the recommended torque levels differ in
the literature. To achieve osseointegration, studies found out
that an insertion torque above 25 and 32 Ncm, respectively,
is necessary [85, 86].
Secondary implant stability
Reverse torque test The reverse torque test measures the crit-
ical torque threshold in which the BIC is destroyed [87]. Thus,
the critical torque threshold indirectly provides information
about the degree of osseointegration, and it has been suggested
that the reverse torque test is a reliable diagnostic method for
measuring osseointegration [88]. On the other hand, this meth-
od has been criticized as destructive and may possibly irrevers-
ibly damage the bony connection between the implant and the
surrounding bone, leading to implant loss [89]. Because the test
only provides information as to an “all or none” outcome, it
cannot quantify the degree of osseointegration and is mainly
361
used in experiments and research [82]. In animal experiments
the animals are sacrificed after a predetermined healing period.
After that, the implants can be removed by applying a counter-
clockwise (reverse) force with a computerized torque driver for
measurement. The reverse torque test was reported to range
from 45 to 48 Ncm. However, it has been speculated that any
reverse torque test greater than 20 Ncm may be acceptable as a
criterion for a successful osseointegration [88].
Resonance frequency analysis The resonance frequency
analysis (RFA) represents a non-invasive diagnostic method
to measure implant stability and bone density at various time
points using vibration and a principle of structural analysis.
RFA utilizes a small L-shaped transducer that is tightened to
the implant or abutment by a screw. The transducer com-
prises of two ceramic elements, one of which is vibrated by
a sinusoidal signal (5–15 kHz), while the other serves as a
receptor. The transducer is screwed directly to the implant
body and shakes the implant at a constant input and ampli-
tude, starting at a low frequency and increasing in pitch until
the implant resonates. Resonance peaks in the received
signal indicate the first flexural resonance frequency of the
implant being measured. Some studies have suggested that
this resonance peak can be used to quantitatively determine
implant stability [75, 89, 90]. The implant possesses suffi-
cient stability is all that can be determined by this method; a
statement regarding the success of a particular implant treat-
ment or the survival rate cannot be made. The implant
stability quotient established by Osstell defines higher im-
plant stability with higher values, and lower values suggest
implant instability [91]. The implant stability quotient can
be fairly reliable if an implant is osseointegrated and if the
implant interface is rigid [92]. In the case of rigid, doubtful
osseointegration, the implant stability quotient tends to fluc-
tuate [93]. The evaluation of implant stability using RFA
still has some uncertain qualities. The method is used clin-
ically without much informative data regarding the bone–
implant interface and resonance frequency values [91].
Therefore, further studies are necessary to increase the reli-
ability of this diagnostic method.
A study about the resonance frequency data and histolog-
ical investigations showed that the resonance frequency data
and histomorphometry did not corellate. Even though a rela-
tive degree of biomechanical fixation was obtained, the RFA
failed to provide detailed information about the inherent me-
chanical properties of the bone–biomaterial interface [9].
Bone–implant contact
Osseointegration is considered to be the crucial criterion for
long-term implant success. The prosthetic denture must form a
functional and firm unit with the osseointegrated implants to
provide resistance to functional and parafunctional loads [94].
362 Oral Maxillofac Surg (2014) 18:355–372

Fig. 5 Example of the evaluation of the bone–implant contact (BIC) surface without bone contact. In “Calculations” (red ellipse), the rela-
with the help of Bioquant Osteo® Software. The green line marks the tive bone implant contact rate is indicated in percentage (%) (bar:
implant surface with bone contact, and the red line marks the implant 5 mm)

Furthermore, it has been shown that successful osseointegra- 24 % after a healing period of 3, 7, 14, 30, and 90 days
tion is directly related to clinically successful long-term [100]. Further preclinical studies investigated implant
results. However, up until now, the amount of an implant osseointegration in combination with a BSM and platelet-
surface that has to be in direct contact with the vital bone rich plasma (PRP) in the porcine frontal skull bone; BIC
(BIC) in order for it to be considered as osseointegrated has ranged from 31 % to 73 % after 2, 4, and 8 weeks [101].
not been determined. It is obvious from the studies above that in vivo studies of
It is assumed that light microscope analysis demonstrates BIC are difficult to compare because of differences in sev-
direct bone contact with the implant and presents the best eral parameters, including the choice of the animal model,
way to quantify BIC and to differentiate between a poten- the anatomical part of the skeleton where the implant is
tially successful osseointegrated device and potentially un- placed, and the age of the animals also has to be taken into
successful osseointegrated implant mainly surrounded by consideration as both bone location and age influence bone
fibrous tissue (Fig. 5). composition [102]. Furthermore, the observed healing peri-
A search of the literature showed that there are more than od differed from 3 days up to 18 months in theses studies, as
800 studies when searching for appropriate in vivo results well as the type, shape, and surface of the implants used.
regarding the amount of BIC needed to define the term Moreover, implant loading influences the amount of bone
“successful osseointegration.” adjacent to the implant surface. Therefore, it remains unclear
For examle, in a preclinical study hollow-cylinder what percentage of BIC is necessary to call an implant
implants with six different surfaces in the metaphyses of successfully integrated, and it remains difficult to transfer
the tibia and femur in miniature pigs were examined [95]. this data to humans. Albrektsson [103] analyzed more than
The BIC was analyzed after a healing period of 3 and 700 consecutively oral implants retrieved from patients and
6 weeks. Depending on the healing period and implant
surface, the BIC ranged from 25 % up to 58 % [95].
Furthermore, the effect of titanium plasma-sprayed implants
with different degrees of roughness on peri-implant bone
formation was investigated [96]. The BIC of implants
placed in the tibial cortical bone of adult goats was 10–
11 % after 3 months. Studies in the mandible of five beagles
showed that the BIC of two different surfaces ranged from
52 % to 78 % after 3 and 6 months [97]. In a histological
and histomorphometric study of human bone specimens,
examinations [98] showed that the addition of autogenous
bone to the BSM Bio-Oss® can produce a high-quality
implant site; 36 implants were uncovered, and the BIC
ranged from 56 % to 72 % after 6 months. Another research
group analyzed the osseointegration of three types of dental
implants in the posterior jaw of adult baboons; the average
BIC for the three different implant surfaces was 64 %, 67 %,
and 70 % following an 18-month evaluation period [99]. Fig. 6 Microfocus computed tomography (micro-CT) image of a
dental implant in the frontal skull of a domestic pig after a healing
Further studies in the frontal skull of the adult domestic pig, period of 30 days. The bone-to-implant contact cannot be measured
comparing a nanotubular implant surface to an untreated around the implant as a consequence of metal artifacts caused by this
microrough surface, showed BIC data ranging from 5 % to investigation method
Oral Maxillofac Surg (2014) 18:355–372
Fig. 7 SEM pictures of the histological specimen. a The interface
between the anodic TiO2 nanotube implant and the bone. A partial
breakage of the interface is due to the histological preparation. b
found that implants placed in the maxilla had an average
BIC of more than 50 % compared to an average BIC of
greater than 75 % in the mandible. Albrektsson suggested
that there has to be more than 50 % hard tissue attachment
for there to be evidence of successful osseointegration of a
dental implant. An interesting observation was that
unloaded implants had a lower BIC, with an average of
10 %, compared to loaded implants [103]. Because of the
wide variety of BIC measured in several studies compared
to Albrektsson investigations, Albrektsson’s defination of
successful osseointegration could not be confirmed. To state
a BIC grade that would guarantee a successful osseointegra-
tion and clinical outcome, further studies are necessary.
Imaging
The common procedure for the representation and morpho-
logic evaluation of bone is conventional X-ray examination,
whereby the picture is determined by the bone structure and
the rate of bone mineralization. Based on the X-ray technique,
numerous methods have been developed that accurately and
automatically measure bone density by measuring X-ray ab-
sorption: X-ray absorptiometry and dual energy X-ray absorp-
tiometry, enable the quantification of BMD [104, 105]. The
difference between these methods is that in dual energy X-ray
absorptiometry, two energetically different X-ray sources are
used simultaneously. Accordingly, different materials can be
differentiated more exactly by using this method in compari-
son to conventional X-ray absorptiometry. Nevertheless, both
Fig. 8 SEM picture of a a
titanium surface and b a
polymer surface
(polyetheretherketone) with
attached osteoblasts after 3 days
of culture. This investigation
method is used to examine the
arrangement, morphology, and
spreading of cells in order to
evaluate material cytotoxicity
363
Magnification of the boxed area in a reveals that the anodic TiO2
nanotubes retain their structure and are not damaged by shearing forces
during the implantation process
procedures offer the possibility of evaluating the bone struc-
ture in vivo.
The use of computed tomography (CT) continues to
grow, although its systematic use in clinical practice has
been limited by concerns about high radiation doses and
relatively high cost [106]. However, CT allows the clinician
to evaluate bone density and other bone tissue parameters on
sectional images. Regeneration and remodeling of the bone
at the implant–tissue interface (secondary stability) can be
determined by this method [107]. According to a clinical
study, CT is an important objective tool for the evaluation of
bone tissue because it enables the clinician to categorize
bone quality as a diagnostic predictor to determine when
to load an implant: immediatelyor later [108].
Besides CT, a new CT method devoted to the imaging of
maxillofacial structures based on the cone beam CT (CBCT)
technique was developed. CBCT allows the implantologist
to acquire 3D volume data in one rotation at reasonably low
levels of radiation dosage. Recently, CT and CBCT tech-
nique were compared to assess which was more reliable.
The superiority of cone beam CT to spiral CT in terms of
spatial resolution on cross-sectional images was confirmed
[109]. Routinely, CBCT data are used for planning of dental
implant insertions or augmentation procedures. Studies rec-
ommend the use of CBCT data for intraforaminal implant
insertions in immediate proximity the mental foramen or the
anterior loop of the inferior alveolar nerve if an adequate
safe distance cannot be maintained, due to clinical reasons
[110]. In the absence of a CBCT scan, safety margin would
364
Fig. 9 Microradiography of
hydroxyapatite BSM in a
monocortical critical size defect
after 2 weeks. a Newly formed
bone from the bottom and sides
of the surrounding bone is
visible. b Bone formation is
partly visible, but complete
osseous integration or any signs
of degradation are not visible
only be achieved with a distance of 6 mm between the
anterior border of the mental foramen and the most distal
interforaminal implant [110]. Several studies showed that
CBCT scans are suitable to detect anatomic variations and
lesions of the maxillary sinus, which are required for dental
implant planning [111, 112]. It could be demonstrated that
using CBCT images, bucco-lingual bone surrounding of
dental implant with a thickness 0.6 mm or more are visually
detectable [113]. It can be stated that CBCT scan represents
a reliable method to detect thin bone plates [113]. However,
an assessment of the accuracy and precision of the CBCT
technique with periapical radiographs (PA) as a reference
showed that PA radiographs should be used as standard
technique to assess interproximal bone level [114]. Using a
standardized PA, crestal bone levels around the implants
could be measured with an accuracy of within 0.2 mm
[115]. Further improvements of hardware and/or software
are required, particularly the artefact problems limits the
accuracy [114].
Also, CT and CBCT offer three-dimensional pictures
and enable statements regarding the structural architecture
of the bone, whereas its small resolving power does not
permit histomorphometric characterization of the individ-
ual trabecula [116, 117].
Therefore, microfocus computed tomography (micro-CT)
has evolved into an interesting tool for the assessment of the
Fig. 10 Histological cross-
section from an implant with a
Bonitex ® coating after a
healing period of 30 days. a
Toludine blue staining; b
microradiography; c
immunohistological staining of
BMP-2 and d osteocalcin
Oral Maxillofac Surg (2014) 18:355–372
three-dimensional architecture of the trabecular bone [118]. A
high-resolution image of the bone structures can be achieved
using this investigation method [119, 120]. Micro-CT is also
used in vivo as a tool for monitoring bone adaptation around
titanium implants [121]. In vivo micro-CT scanning method-
ology makes it possible to perform longitudinal studies in
small animals, following bone changes over time, while re-
ducing the number of animals needed to assess different time
points using conventional histology [122]. On the other hand,
micro-CT does have drawbacks, such as metal artifacts caused
by a combination of beam hardening, scatter, and nonlinear
partial volume effect [123] (Fig. 6).
As a consequence, bone contact cannot be measured around
implants with metallic surfaces on micro-CT slices [124].
Another limitation is a maximum local dissolution (reso-
lution) of 2–30 μm and an acquisition time of several hours
per sample. Therefore, this method is only useful for ana-
lyzing the microstructure of research samples [125]. A clin-
ical use on the patient is not possible due to the
overestimation of bone volume and is influenced by the
amount of osteoid. However, in a direct comparison with
histomorphometrically evaluated bone specimens, the de-
scription of trabecular and marrow space provided by
micro-CT was more accurate [126].
Unfortunately, the micro-CT technique depends on the
mineralisation rate of the bone tissue. Thereby, newly
Oral Maxillofac Surg (2014) 18:355–372
Fig. 11 Toluidine blue staining of histological cross-sections from
three different bone substitution materials. a BioOss, a natural bovine
hydroxyapatite; b PepGen P-15, a calcium phosphate matrix combined
formed bone, which is not mineralised in the early phases of
bone formation, cannot be quantified through this method.
Quantification assesement of newly formed bone, histolog-
ical sections remain the gold standard due to better resolu-
tion compared to the micro-CT technique.
For the morphological characterization of sample surfa-
ces in vivo, field emission scanning electron microscopy can
also be used. With this technique, the surface of the implant
as well the surrounding bone and bone–implant interface
can be qualitatively analyzed (Fig. 7), and it users offers the
opportunity to evaluate the condition of an implant coating
after implant insertion.
Using a gold–palladium target for the sputter coating,
even structures in the nanometer range can be detected and
evaluated [127]. Yet, the complexity of sample preparation
does not make field emission scanning electron microscopy
a routinely used method in experimental in vivo research
compared to in vitro studies (Fig. 8).
Microradiography
An important quantitative imaging method for the histomor-
phometric evaluation of bone is microradiography, which is
Fig. 12 Paraffin cross-sections of bone samples stained using Mas-
son's trichrome stain. This staining method allows for qualitative
examination of pathological changes in the bone structure. a The bone
of the healthy control animal. A regular structure with areas of phys-
iological bone remodeling is visble. b Bone samples from diabetic
365
with a synthetically produced amino acid sequence (P-15); and c
Ostim, a synthetic hydroxyapatite at 12 weeks in a critical size defect
in the frontal skull of an adult domestic pig
commonly used to measure mineral distribution in bone and
bone regeneration, providing high-resolution radiographs of
bone growth in a bony defect [128, 129]. Microradiography
was first used to histomorphometrically investigate bone
samples in the 1950s [130]. This method also enables the
quantitative evaluation of bone growth and bone formation
on implant surfaces and the biological behavior of BSM in
vivo [131] (Figs. 9 and 10b).
For microradiographic examination, embedded bone sec-
tions, 150–180 μm thick, are produced by the saw and
grinding technique [132]. The equipment consists of an X-
ray generator and a lensless camera. Depending on the
sample material, a special film is exposed to different energy
levels for different exposure times, producing a micro ra-
diograph. The contrast of the picture is determined by the
mineralization rate of the investigated samples [133]. The
following histomorphometric parameters based on the plate-
like trabecular model can be evaluated: the bone minerali-
zation rate of the newly formed bone; the trabecular bone
volume (BV/TV; %); the trabecular bone pattern factor
(TBPf), which describes the extent of intertrabecular con-
nectivity. The intertrabecular connectivity increases with
decreasing TBPf [134].
animals. Wider and irregularly shaped trabecular bones are visible.
The mineralization zones are visualized as red-stained bone areas and
are expanded compared to the mineralization zones the healthy control
animal`s bone, indicating a higher rate of bone remodeling
366 Oral Maxillofac Surg (2014) 18:355–372

Fig. 13 Alizarin Red S staining

is used to quantification bone
formation and mineralized
bone. The pictures show dental
implants and the surrounding
connetive tissue. The
mineralized regions containing
Calcium, form an Alizarin Red–
S calcium complex in a
chelation process. The calcium
deposits are stained orange-red
[155]

Light microscopy Masson's trichrome stain This staining method represents

today's standard coloring in histomorphometry. In addition
Although information concerning the relative degree of to good cell staining, mineralized and non-mineralized bone
biomechanical fixation is provided by mechanical tests, matrix can also be well differentiated. Mineralized bone ma-
and the bone formation can be estimated by X-ray analysis, trix and collagen are stained a bright green, calcified cartilage
these tests do not provide detailed microscopic information matrix I light green, osteoid red, cell cores blue, cytoplasm
about the inherent mechanical properties of the bone–bio- reddish-brown, and erythrocytes orange. This stain is com-
material interface. Light microscopy allows for a qualitative monly used to qualitatively evaluate bone (Fig. 12).
statement concerning tissue reactions and regeneration char-
acteristics by different staining methods. Alizarin Red-S stain Osteoblasts build bone-matrix proteins,
For bonesthe following parameters are commonly evaluat- which are able to incorporate calciumphosphate. Alizarin Red-
ed in biomaterial and implant research: the trabecular bone
volume (BV/TV; %); the buccal and oral cortical bone volume
(¼ cortical bone volume per tissue volume; %); the TBPf; and
the BIC, which determines the percentage of direct contact
between mineralized bone and the implant surface.
Four of the most common stainings used for the micro-
scopic evaluation of bone–implant interactions, bone forma-
tion and bone quality are Toluidine blue, Masson's
trichrome, Alizarin Red-S and von Kossa staining.

Toluidine blue In this specific staining, mineralized lami-

nated tissue presents as uncolored to pale blue; cells, cell
cores, osteoid fringes, cement lines, collagen fibers, and soft
tissue colors are notably blue; cartilage matrix and early
wound healing areas are metachromatic red-violet; and cal-
cified matrix is dark blue [132] (Figs. 1, 10a and 11).
Fig. 14 In this picture, von Kossa staining is used to quantify miner-
alized areas of collagenous matrix. During staining procedures, silver/
calcium complexes ocurre in the mineralized areas. Using light illumi-
nation, the stained areas have a black color
Oral Maxillofac Surg (2014) 18:355–372
Fig. 15 Regions of interest (ROI) for the histological evaluation of the
bone–implant contact (BIC) and quantitative immunohistochemical
analysis of Osteocalcin. The dashed line shows the dimensions of the
removed implant
S is used to quantitatively determine the presence of calcific
depositions in extracellular matrix of tissue sections and oste-
oblastic cell lines [135, 136]. Calcium forms an Alizarin Red
S–calcium complex in a chelation process. The reaction is
birefringent. Thus, Alizarin Red-S stain is qualified to describe
cells of an osteogenic lineage and represents a marker of matrix
mineralization which is associated with bone formation. The
calcium deposits are stained orange-red (Fig. 13).
von Kossa stain von Kossa staining is used to quantify min-
eralization in cell culture and tissue sections [137–139]. The
staining principle is a precipitation reaction. Thereby silver
ions react with phosphate in the presence of acidic material
and build complexes in the mineralized areas. Photochemical
degradation of silver phosphate to silver occurs under light
illumination. The stained areas have a black color (Fig. 14).
Fig. 16 a Toluidine blue
staining and b fluorescence
microscopic image of bovine
hydroxyapatite (BioOss®)
4 weeks afer implantation.
Osseointegration of the BSM is
complete and the sequence of
bone growth is after the
application of intravital
fluorochromes
367
Immunohistochemistry
Immunohistochemistry refers to the process of localizing
proteins in the cells of a tissue section, exploiting the prin-
ciple of specific antibody binding to antigens in biological
tissues. Specific molecular markers are characteristic of
particular cellular events, such as proliferation or apoptosis.
Immunohistochemistry is also widely used in basic research
to understand the distribution and localization of biomarkers
and differentially expressed proteins in different parts of a
biological tissue. Due to immunohistochemistry, it is possi-
ble to identify multiple cytokines that affect all stages of de
novo bone formation (Fig. 2). Knowing the cytokine ex-
pression makes it possible to evaluate the time sequence of
the bone regeneration process [140, 141]. The bone mor-
phogenetic proteins (BMPs) are a family of cytokines that
stimulate the proliferation of chondrocytes and osteoblasts.
These proteins are morphogenic and induce MSC differen-
tiation into osteoblasts [142] (Fig. 10c). Collagen type I is
considered to be a basic initial bone matrix protein in bone
formation [143]. The collagen forms a scaffold for cell
attachment and migration in tissues and modulates cell
differentiation and morphogenesis by mediating the flux of
chemical and mechanical stimuli [144]. During skeletal
structure remodeling, reparative cells migrate on this matrix
and subsequently organize and remodel the matrix through
cytoskeleton and matrix synthesis and degradation [145].
The expression of collagen type I is described as an early
indicator of de novo bone formation and can be identified
and quantified by immunohistochemistry in order to show
new bone formation around the implants.
A late marker of bone development that is often investi-
gated due to its delayed appearance in bone formation and
development is osteocalcin [146] (Fig. 10d). This molecule
is a small non-collagenous protein with a molecular weight
of 5,800 kDa. Osteocalcin is synthesized and secreted dur-
ing osteoblast differentiation, and mineralization is a unique
product of osteoblasts and odontoblasts [147, 148]. Osteo-
calcin is the most abundant non-collagenous protein in bone
and can identified by immunhistochemical methods. Most
of the protein is incorporated into the bone matrix, where it
368
Fig. 17 Fluorescence microscopic image labeled with oxytetracycline
(*application day 7), alizarin complex (# application day 14), and
xylenol orange (†application day 46) to evaluate the time-dependent
peri-implant bone formation after a healing period of 8 weeks
is bound by hydroxyapatite, and its appearance during bone
maturation is delayed by collagen type I and indicates the
mineralization process of bone formation implemented by
osteocyte calcification.
In order to quantitatively asses the expression of bone
matrix proteins, computer programs, such as Bioquant
Osteo software V7.10.10 (Bioquant Image Analysis Coop-
eration, Nashville, TN), are used. Using this program, the
stained area of an immunohistochemical sample can be
determined and calculated as a percentage of the total area
[149] (Fig. 15).
Fluorescence
The temporal operational sequence of bone growth, change,
and healing is portrayed imperfectly using conventional his-
tological techniques. However, intravital fluorochrome can
open up the dynamics of bone formation processes (Fig. 16).
The area of active mineralization can be labeled by ap-
plying calcium-chelating fluorescent dyes [150, 151]. These
substances are inserted during bone formation in vivo and
are later visible due to their fluorescence. This histological
investigation method offers a more precise evaluation of the
morphological characteristics of the bone architecture [152].
The administration of fluorescent dyes allows one to follow
the direction and topographic localization of new bone
formation (Fig. 17).
During the mineralization process, the fluorescent dyes
are incorporated at the mineralization front by chelation
[153]. In bone, the technique is used to investigate biolog-
ical rhythms, rates of mineralization, chronological devel-
opment, and an individual bone´s response to external
influences [9]. Commonly used agents that can be differen-
tiated due to their special emission spectra are oxytetracy-
cline, alizarin complex, calcein blue, and xylenol orange [9,
149]. These dyes are usually applied via intramuscular
Oral Maxillofac Surg (2014) 18:355–372
injections in 2 % NaHCO3 solution. After sacrifice of the
animal, the specimens are grounded to about 20–30 μm for
fluorescence microscopy by utilizing the “sawing and grind-
ing” technique, which allows the quantification of bone
formation during the period of dye application. [154].
Acknowledgements We would like to thank Dr. Jochen Zwerina,
Department of Internal Medicine 3 and Institute for Clinical Immunol-
ogy, University of Erlangen–Nuremberg, for the von Kossa bone
sections.
References
1. Kammula R, Morris J (2001) Considerations for the biocompat-
ibility evaluation of medical devices. Med Device Diagn Ind.
http://www.mddionline.com
2. DFG DF (1984) Der Tierversuch als wissenschaftliche Methode.
In: Tierexperimentelle Forschung und Tierschutz. Mitteilung III
der Kommission für Versuchstierforschung. VCH Verlagsgesell-
schaft mbH, Weinheim, pp 17–22
3. Zutphen LV, Baumans V, Beynen A (1995) Grundlagen der
Versuchstierkunde. Verlag GF, Stuttgart
4. Held J (1983) Appropriate animal models. The role of animals in
biomedical research. Ann N Y Acad Sci 406:13–19
5. Grünberg W (1992) Der Tierversuch als Methode der biomedizi-
nischen Forschung. In: Kronberger L (ed) Experimentelle Chir-
urgie. Ferdinand Enke Verlag, Stuttgart, pp 18–24
6. Nunamaker DM (1998 Oct) Experimental models of fracture
repair. Clin Orthop Relat Res (355 Suppl):S56–65
7. Bosetti M, Zanardi L, Hench L, Cannas M (2003) Type I collagen
production by osteoblast-like cells cultured in contact with dif-
ferent bioactive glasses. J Biomed Mater Res A 64(1):189–195
8. Metak G, Gomoll A, Wolter W, Barth G, Ascherl R (1998)
Interspecies comparison of healing standardazed bone defects
with and without autogenous bone transplantation. Langenbecks
Arch Chir Suppl Kongressbd 115(Suppl I):25–30
9. Nkenke E, Hahn M, Weinzierl K, Radespiel-Troger M, Neukam
FW, Engelke K (2003) Implant stability and histomorphometry: a
correlation study in human cadavers using stepped cylinder
implants. Clin Oral Implants Res 14(5):601–609
10. Nkenke E, Kloss F, Wiltfang J, Schultze-Mosgau S, Radespiel-
Troger M, Loos K et al (2002) Histomorphometric and fluores-
cence microscopic analysis of bone remodelling after installation
of implants using an osteotome technique. Clin Oral Implants Res
13(6):595–602
11. Rupprecht S, Bloch-Birkholz A, Lethaus B, Rosiwal S, Neukam
FW, Schlegel A (2005) The bone–metal interface of defect and
press-fit ingrowth of microwave plasma-chemical vapor deposition
implants in the rabbit model. Clin Oral Implants Res 16(1):98–104
12. Administration UFaD (2005) 510(k) premarket notification
#K041177 for SYNTHACER and SYNTRICER according to
regulation number 888.3045. Administration UFaD, Rockville
13. Gilsanz V, Roe TF, Gibbens DT, Schulz EE, Carlson ME, Gonzalez
O et al (1988) Effect of sex steroids on peak bone density of
growing rabbits. Am J Physiol 255(4 Pt 1):E416–E421
14. Wang X, Mabrey JD, Agrawal CM (1998) An interspecies com-
parison of bone fracture properties. Biomed Mater Eng 8(1):1–9
15. Sturmer KM, Schuchardt W (1980) New aspects of closed intra-
medullary nailing and marrow cavity reaming in animal experi-
ments: I. The tibia of the sheep, as a model for intramedullar
nailing (author's transl). Unfallheilkunde 83(7):341–345
Oral Maxillofac Surg (2014) 18:355–372
16. Vignoletti F, Abrahamsson I (2012) Quality of reporting of ex-
perimental research in implant dentistry. Critical aspects in de-
sign, outcome assessment and model validation. J Clin
Periodontol 39(Suppl 12):6–27
17. Marshall M, Oberhofer H, Staubesand J (1980) Early micro- and
macro-angiopathy in the streptozotocin diabetic minipig. Res Exp
Med (Berl) 177(2):145–158
18. Laiblin C, Jaeschke G (1979) Clinical chemistry examinations of
bone and muscle metabolism under stress in the Gottingen min-
iature pig—an experimental study. Berl Munch Tierarztl
Wochenschr 92(6):124–128
19. Beddoe AH (1978) A quantitative study of the structure of
trabecular bone in man, rhesus monkey, beagle and miniature
pig. Calcif Tissue Res 25(3):273–281
20. Swindle MM, Smith AC, Hepburn BJ (1988) Swine as models in
experimental surgery. J Invest Surg 1(1):65–79
21. Eitel F, Klapp F, Jacobson W, Schweiberer L (1981) Bone regen-
eration in animals and in man. A contribution to understanding
the relative value of animal experiments to human pathophysiol-
ogy. Arch Orthop Trauma Surg 99(1):59–64
22. Schlegel KA, Lang FJ, Donath K, Kulow JT, Wiltfang J (2006)
The monocortical critical size bone defect as an alternative ex-
perimental model in testing bone substitute materials. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 102(1):7–13
23. Aerssens J, Boonen S, Lowet G, Dequeker J (1998) Interspe-
cies differences in bone composition, density, and quality:
potential implications for in vivo bone research. Endocrinolo-
gy 139(2):663–670
24. Park J, Lutz R, Felszeghy E, Wiltfang J, Nkenke E, Neukam FW
et al (2007) The effect on bone regeneration of a liposomal vector
to deliver BMP-2 gene to bone grafts in peri-implant bone
defects. Biomaterials 28(17):2772–2782
25. Lutz R, Srour S, Nonhoff J, Weisel T, Damien CJ, Schlegel KA
(2010) Biofunctionalization of titanium implants with a biomi-
metic active peptide (P-15) promotes early osseointegration. Clin
Oral Implants Res 21(7):726–734
26. Schlegel KA, Thorwarth M, Plesinac A, Wiltfang J, Rupprecht S
(2006) Expression of bone matrix proteins during the osseus
healing of topical conditioned implants: an experimental study.
Clin Oral Implants Res 17(6):666–672
27. Wehrhan F, Amann K, Molenberg A, Lutz R, Neukam FW,
Schlegel KA (2012) PEG matrix enables cell-mediated local
BMP-2 gene delivery and increased bone formation in a porcine
critical size defect model of craniofacial bone regeneration. Clin
Oral Implants Res 23(7):805–813
28. Schlegel KA, Rupprecht S, Petrovic L, Honert C, Srour S, von
Wilmowsky C et al (2009) Preclinical animal model for de novo
bone formation in human maxillary sinus. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 108(3):e37–e44
29. Phol Y, Joos G, at al (1995) Der Hund als Modell für die
zahnärztliche Implantologie. Acta Chir. Austriaca Supplement
117
30. Gong J, Arnold J, Cohn S (1964) Composition of trabecular and
cortical bone. Anat Rec 149:325–332
31. Kimmel DB, Jee WS (1982) A quantitative histologic study of
bone turnover in young adult beagles. Anat Rec 203(1):31–45
32. Finlay JB, Hurtig MB, Hardie WR, Liggins AB, Batte SW (1995)
Geometrical properties of the ovine tibia: a suitable animal model
to study the pin–bone interface in fracture fixation? Proc Inst
Mech Eng H 209(1):37–50
33. deKleer V (2006) Development of bone. In: Sumner-Smith G
(ed) Bone in clinical orthopaedics. Saunders Co, Philadelphia, pp
1–80
34. Nafei A, Kabel J, Odgaard A, Linde F, Hvid I (2000) Properties
of growing trabecular ovine bone: Part II. Architectural and
mechanical properties. J Bone Joint Surg Br 82(6):921–927
369
35. Liebschner MA (2004) Biomechanical considerations of animal
models used in tissue engineering of bone. Biomaterials
25(9):1697–1714
36. Turner AS (2001) Animal models of osteoporosis—necessity and
limitations. Eur Cell Mater 1:66–81
37. Nafei A, Danielsen CC, Linde F, Hvid I (2000) Properties of
growing trabecular ovine bone: Part I. Mechanical and physical
properties. J Bone Joint Surg Br 82(6):910–920
38. Knauf M, Gerds T, Muche R, Strub JR (2007) Survival and
success rates of 3i implants in partially edentulous patients:
results of a prospective study with up to 84-months' follow-up.
Quintessence Int 38(8):643–651
39. Newman J, Pydisetty RV, Ackroyd C (2009) Unicompartmental
or total knee replacement: the 15-year results of a prospective
randomised controlled trial. J Bone Joint Surg Br 91(1):52–57
40. Baumann B, Hendrich C, Barthel T, Bockholt M, Walther M,
Eulert J et al (2007) 9- to 11-year results of cemented titanium
mueller straight stem in total hip arthroplasty. Orthopedics
30(7):551–557
41. Beikler T, Flemmig TF (2003) Implants in the medically com-
promised patient. Crit Rev Oral Biol Med 14(4):305–316
42. Wild S, Roglic G, Green A, Sicree R, King H (2004) Global
prevalence of diabetes: estimates for the year 2000 and projec-
tions for 2030. Diabetes Care 27(5):1047–1053
43. Mellado-Valero A, Ferrer Garcia JC, Herrera Ballester A, Labaig
Rueda C (2007) Effects of diabetes on the osseointegration of
dental implants. Med Oral Patol Oral Cir Bucal 12(1):E38–E43
44. Phalen RF, Mannix RC, Drew RT (1984) Inhalation exposure
methodology. Environ Health Perspect 56:23–34
45. Marshall M (1979) Induction of chronic diabetes by streptozoto-
cin in the miniature pig. Res Exp Med (Berl) 175(2):187–196
46. Muggenburg BA, Tilley L, Green FH (2000) Animal models of
cardiac disease: potential usefulness for studying health effects of
inhaled particles. Inhal Toxicol 12(9):901–925
47. von Wilmowsky C, Stockmann P, Metzler P, Harsch IA, Amann
K, Schlegel KA (2010) Establishment of a streptozotocin-induced
diabetic domestic pig model and a systematic evaluation of path-
ological changes in the hard and soft tissue over a 12-month
period. Clin Oral Implants Res 21(7):709–717
48. Schlegel KA, Prechtl C, Most T, Seidl C, Lutz R, von Wilmowsky
C (2011 Nov 24) Osseointegration of SLActive implants in diabetic
pigs. Clin Oral Implants Res
49. von Wilmowsky C, Stockmann P, Harsch I, Amann K, Metzler P,
Lutz R et al (2011) Diabetes mellitus negatively affects peri-
implant bone formation in the diabetic domestic pig. J Clin
Periodontol 38(8):771–779
50. Barlet JP, Coxam V, Davicco MJ, Gaumet N (1994) Animal
models of post-menopausal osteoporosis. Reprod Nutr Dev
34(3):221–236
51. Zarrinkalam MR, Beard H, Schultz CG, Moore RJ (2009) Vali-
dation of the sheep as a large animal model for the study of
vertebral osteoporosis. Eur Spine J 18(2):244–253
52. Zarrinkalam MR, Mulaibrahimovic A, Atkins GJ, Moore RJ
(2011 May 31) Changes in osteocyte density correspond with
changes in osteoblast and osteoclast activity in an osteoporotic
sheep model. Osteoporos Int
53. Egermann M, Goldhahn J, Holz R, Schneider E, Lill CA (2008)
A sheep model for fracture treatment in osteoporosis: benefits of
the model versus animal welfare. Lab Anim 42(4):453–464
54. Goldhahn J, Jenet A, Schneider E, Lill CA (2005) Slow rebound
of cancellous bone after mainly steroid-induced osteoporosis in
ovariectomized sheep. J Orthop Trauma 19(1):23–28
55. Lill CA, Gerlach UV, Eckhardt C, Goldhahn J, Schneider E
(2002) Bone changes due to glucocorticoid application in an
ovariectomized animal model for fracture treatment in osteopo-
rosis. Osteoporos Int 13(5):407–414
370
56. Ding M, Danielsen CC, Overgaard S (2011 Jul 13) The effects of
glucocorticoid on microarchitecture, collagen, mineral and mechan-
ical properties of sheep femur cortical bone. J Tissue Eng Regen Med
57. Fini M, Giardino R, Borsari V, Torricelli P, Rimondini L, Giavaresi
G et al (2003) In vitro behaviour of osteoblasts cultured on ortho-
paedic biomaterials with different surface roughness, uncoated and
fluorohydroxyapatite-coated, relative to the in vivo osteointegration
rate. Int J Artif Organs 26(6):520–528
58. Guchelaar HJ, Vermes A, Meerwaldt JH (1997) Radiation-
induced xerostomia: pathophysiology, clinical course and sup-
portive treatment. Support Care Cancer 5(4):281–288
59. Ihde S, Kopp S, Gundlach K, Konstantinovic VS (2009) Effects
of radiation therapy on craniofacial and dental implants: a review
of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 107(1):56–65
60. Brogniez V, Nyssen-Behets C, Gregoire V, Reychler H, Lengele
B (2002) Implant osseointegration in the irradiated mandible. A
comparative study in dogs with a microradiographic and histo-
logic assessment. Clin Oral Implants Res 13(3):234–242
61. Johnsson AA, Sawaii T, Jacobsson M, Granstrom G, Turesson I
(2000) A histomorphometric and biomechanical study of the
effect of delayed titanium implant placement in irradiated rabbit
bone. Clin Implant Dent Relat Res 2(1):42–49
62. Asikainen P, Klemetti E, Kotilainen R, Vuillemin T, Sutter F,
Voipio HM et al (1998) Osseointegration of dental implants in
bone irradiated with 40, 50 or 60 Gy doses. An experimental
study with beagle dogs. Clin Oral Implants Res 9(1):20–25
63. Schon R, Ohno K, Kudo M, Michi K (1996) Peri-implant tissue
reaction in bone irradiated the fifth day after implantation in
rabbits: histologic and histomorphometric measurements. Int J
Oral Maxillofac Implants 11(2):228–238
64. Verdonck HW, Meijer GJ, Laurin T, Nieman FH, Stoll C, Riediger
D et al (2008) Implant stability during osseointegration in irradiated
and non-irradiated minipig alveolar bone: an experimental study.
Clin Oral Implants Res 19(2):201–206
65. Thorn JJ, Hansen HS, Specht L, Bastholt L (2000) Osteoradio-
necrosis of the jaws: clinical characteristics and relation to the
field of irradiation. J Oral Maxillofac Surg 58(10):1088–1093,
discussion 93–5
66. Xu J, Zheng Z, Fang D, Gao R, Liu Y, Fan ZP et al (2012) Early-
stage pathogenic sequence of jaw osteoradionecrosis in vivo. J
Dent Res 91(7):702–708
67. Tamplen M, Trapp K, Nishimura I, Armin B, Steinberg M,
Beumer J et al (2011) Standardized analysis of mandibular
osteoradionecrosis in a rat model. Otolaryngol Head Neck Surg
145(3):404–410
68. Fenner M, Park J, Schulz N, Amann K, Grabenbauer GG, Fahrig
A et al (2010) Validation of histologic changes induced by exter-
nal irradiation in mandibular bone. An experimental animal mod-
el. J Craniomaxillofac Surg 38(1):47–53
69. Zhang WB, Zheng LW, Chua D, Cheung LK (2010) Bone regen-
eration after radiotherapy in an animal model. J Oral Maxillofac
Surg 68(11):2802–2809
70. Colella G, Cannavale R, Pentenero M, Gandolfo S (2007) Oral
implants in radiated patients: a systematic review. Int J Oral
Maxillofac Implants 22(4):616–622
71. Yerit KC, Posch M, Seemann M, Hainich S, Dortbudak O,
Turhani D et al (2006) Implant survival in mandibles of irradiated
oral cancer patients. Clin Oral Implants Res 17(3):337–344
72. Liddelow G, Klineberg I (2011) Patient-related risk factors for
implant therapy. A critique of pertinent literature. Aust Dent J
56(4):417–426, quiz 41
73. Branemark PI, Hansson BO, Adell R, Breine U, Lindstrom J,
Hallen O et al (1977) Osseointegrated implants in the treatment of
the edentulous jaw. Experience from a 10-year period. Scand J
Plast Reconstr Surg Suppl 16:1–132
Oral Maxillofac Surg (2014) 18:355–372
74. Roberts WE (1988) Bone tissue interface. J Dent Educ
52(12):804–809
75. Meredith N (1998) Assessment of implant stability as a prognos-
tic determinant. Int J Prosthodont 11(5):491–501
76. Davies JE (1998) Mechanisms of endosseous integration. Int J
Prosthodont 11(5):391–401
77. Raghavendra S, Wood MC, Taylor TD (2005) Early wound
healing around endosseous implants: a review of the literature.
Int J Oral Maxillofac Implants 20(3):425–431
78. Sennerby L, Roos J (1998) Surgical determinants of clinical
success of osseointegrated oral implants: a review of the litera-
ture. Int J Prosthodont 11(5):408–420
79. Cochran DL, Schenk RK, Lussi A, Higginbottom FL, Buser D
(1998) Bone response to unloaded and loaded titanium implants
with a sandblasted and acid-etched surface: a histometric study in
the canine mandible. J Biomed Mater Res 40(1):1–11
80. Degidi M, Daprile G, Piattelli A (2010) Determination of primary
stability: a comparison of the surgeon's perception and objective
measurements. Int J Oral Maxillofac Implants 25(3):558–561
81. Friberg B, Sennerby L, Grondahl K, Bergstrom C, Back T,
Lekholm U (1999) On cutting torque measurements during im-
plant placement: a 3-year clinical prospective study. Clin Implant
Dent Relat Res 1(2):75–83
82. Atsumi M, Park SH, Wang HL (2007) Methods used to assess
implant stability: current status. Int J Oral Maxillofac Implants
22(5):743–754
83. Liu C, Tsai MT, Huang HL, Chen MY, Hsu JT, Su KC, et al (2012
Jan 10) Relation between insertion torque and bone–implant
contact percentage: an artificial bone study. Clin Oral Investig
84. Cho KC, Baek SH (2011 Nov 3) Effects of predrilling depth and
implant shape on the mechanical properties of orthodontic mini-
implants during the insertion procedure. Angle Orthod
85. Norton MR (2011) The influence of insertion torque on the
survival of immediately placed and restored single-tooth
implants. Int J Oral Maxillofac Implants 26(6):1333–1343
86. Ottoni JM, Oliveira ZF, Mansini R, Cabral AM (2005) Correla-
tion between placement torque and survival of single-tooth
implants. Int J Oral Maxillofac Implants 20(5):769–776
87. Johansson C, Albrektsson T (1987) Integration of screw implants
in the rabbit: a 1-year follow-up of removal torque of titanium
implants. Int J Oral Maxillofac Implants 2(2):69–75
88. Sullivan DY, Sherwood RL, Collins TA, Krogh PH (1996) The
reverse-torque test: a clinical report. Int J Oral Maxillofac
Implants 11(2):179–185
89. Meredith N (1998) A review of nondestructive test methods and
their application to measure the stability and osseointegration of
bone anchored endosseous implants. Crit Rev Biomed Eng
26(4):275–291
90. Sennerby L, Meredith N (2008) Implant stability measurements
using resonance frequency analysis: biological and biomechani-
cal aspects and clinical implications. Periodontol 47:51–66
91. Barewal RM, Oates TW, Meredith N, Cochran DL (2003) Reso-
nance frequency measurement of implant stability in vivo on
implants with a sandblasted and acid-etched surface. Int J Oral
Maxillofac Implants 18(5):641–651
92. Nedir R, Bischof M, Szmukler-Moncler S, Bernard JP, Samson J
(2004) Predicting osseointegration by means of implant primary
stability. Clin Oral Implants Res 15(5):520–528
93. Friberg B, Sennerby L, Linden B, Grondahl K, Lekholm U
(1999) Stability measurements of one-stage Branemark implants
during healing in mandibles. A clinical resonance frequency
analysis study. Int J Oral Maxillofac Surg 28(4):266–272
94. Skalak R (1983) Biomechanical considerations in osseointegrated
prostheses. J Prosthet Dent 49(6):843–848
95. Buser D, Schenk RK, Steinemann S, Fiorellini JP, Fox CH, Stich
H (1991) Influence of surface characteristics on bone integration
Oral Maxillofac Surg (2014) 18:355–372
of titanium implants. A histomorphometric study in miniature
pigs. J Biomed Mater Res 25(7):889–902
96. Vercaigne S, Wolke JG, Naert I, Jansen JA (1998) Histomorpho-
metrical and mechanical evaluation of titanium plasma-spray-
coated implants placed in the cortical bone of goats. J Biomed
Mater Res 41(1):41–48
97. Abrahamsson I, Zitzmann NU, Berglundh T, Wennerberg A,
Lindhe J (2001) Bone and soft tissue integration to titanium
implants with different surface topography: an experimental
study in the dog. Int J Oral Maxillofac Implants 16(3):323–332
98. Yildirim M, Spiekermann H, Handt S, Edelhoff D (2001) Max-
illary sinus augmentation with the xenograft Bio-Oss and autog-
enous intraoral bone for qualitative improvement of the implant
site: a histologic and histomorphometric clinical study in humans.
Int J Oral Maxillofac Implants 16(1):23–33
99. Watzak G, Zechner W, Ulm C, Tangl S, Tepper G, Watzek G
(2005) Histologic and histomorphometric analysis of three
types of dental implants following 18 months of occlusal
loading: a preliminary study in baboons. Clin Oral Implants
Res 16(4):408–416
100. von Wilmowsky C, Bauer S, Lutz R, Meisel M, Neukam FW,
Toyoshima T et al (2009) In vivo evaluation of anodic TiO2
nanotubes: an experimental study in the pig. J Biomed Mater
Res B Appl Biomater 89(1):165–171
101. Schlegel KA, Kloss FR, Kessler P, Schultze-Mosgau S, Nkenke
E, Wiltfang J (2003) Bone conditioning to enhance implant
osseointegration: an experimental study in pigs. Int J Oral Max-
illofac Implants 18(4):505–511
102. Aerssens J, Boonen S, Joly J, Dequeker J (1997) Variations in
trabecular bone composition with anatomical site and age: poten-
tial implications for bone quality assessment. J Endocrinol
155(3):411–421
103. Albrektsson T (2008) Hard tissue implant interface. Aust Dent J
53(Suppl 1):S34–S38
104. Qin M, Lin S, Song Z, Tian J, Chen F, Yan H et al (1999)
Comparison of bone mass in forearm, lumbar vertebra and hip
by single and/or dual energy X-ray absorptiometry. Chin Med Sci
J 14(2):117–120
105. Cherian RA, Haddaway MJ, Davie MW, McCall IW, Cassar-
Pullicino VN (2000) Effect of Paget's disease of bone on areal
lumbar spine bone mineral density measured by DXA, and den-
sity of cortical and trabecular bone measured by quantitative CT.
Br J Radiol 73(871):720–726
106. Frederiksen NL (1995) Diagnostic imaging in dental implantol-
ogy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
80(5):540–554
107. Myoung H, Kim YY, Heo MS, Lee SS, Choi SC, Kim MJ (2001)
Comparative radiologic study of bone density and cortical thick-
ness of donor bone used in mandibular reconstruction. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod 92(1):23–29
108. Ericsson I, Nilner K (2002) Early functional loading using Brane-
mark dental implants. Int J Periodontics Restorative Dent
22(1):9–19
109. Kobayashi K, Shimoda S, Nakagawa Y, Yamamoto A (2004)
Accuracy in measurement of distance using limited cone-beam
computerized tomography. Int J Oral Maxillofac Implants
19(2):228–231
110. Apostolakis D, Brown JE (2012) The anterior loop of the inferior
alveolar nerve: prevalence, measurement of its length and a
recommendation for interforaminal implant installation based on
cone beam CT imaging. Clin Oral Implants Res 23(9):1022–1030
111. Pelinsari Lana J, Moura Rodrigues Carneiro P, de Carvalho
Machado V, Eduardo Alencar de Souza P, Ricardo Manzi F,
Campolina Rebello Horta M (2011 Oct 3) Anatomic variations
and lesions of the maxillary sinus detected in cone beam com-
puted tomography for dental implants. Clin Oral Implants Res
371
112. Naitoh M, Suenaga Y, Gotoh K, Ito M, Kondo S, Ariji E (2010)
Observation of maxillary sinus septa and bony bridges using dry
skulls between Hellman's dental age of IA and IIIC. Okajimas
Folia Anat Jpn 87(2):41–47
113. Naitoh M, Hayashi H, Tsukamoto N, Ariji E (2012) Labial bone
assessment surrounding dental implant using cone-beam comput-
ed tomography: an in vitro study. Clin Oral Implants Res
23(8):970–974
114. Raes F, Renckens L, Aps J, Cosyn J, De Bruyn H (2011 Oct 18)
Reliability of circumferential bone level assessment around single
implants in healed ridges and extraction sockets using cone beam
CT. Clin Implant Dent Relat Res
115. Hermann JS, Schoolfield JD, Nummikoski PV, Buser D, Schenk
RK, Cochran DL (2001) Crestal bone changes around titanium
implants: a methodologic study comparing linear radiographic
with histometric measurements. Int J Oral Maxillofac Implants
16(4):475–485
116. Prevrhal S, Genant HK (1999) Quantitative computer tomogra-
phy. Radiologe 39(3):194–202
117. Reichel H, Lebek S, Alter C, Hein W (1998) Biomechanical and
densitometric bone properties after callus distraction in sheep.
Clin Orthop Relat Res 357:237–246
118. Müller R (2002) The Zürich experience. One decade of threedi-
mensional high-resolution computed tomography. Top Magn
Reson Imaging 13:307–322
119. Ruegsegger P, Koller B, Muller R (1996) A microtomographic
system for the nondestructive evaluation of bone architecture.
Calcif Tissue Int 58(1):24–29
120. Muller R, Hildebrand T, Ruegsegger P (1994) Non-invasive bone
biopsy: a new method to analyse and display the three-dimensional
structure of trabecular bone. Phys Med Biol 39(1):145–164
121. Holdsworth D, Thornton M (2002) Micro-CT in small animal and
specimen imaging. Trends Biotechnol 20:34–39
122. Smet ED, Jaecques S, Wevers M, Jansen J, Jacobs R, Sloten J et
al (2006) Effect of controlled early implant loading on bone
healing and bone mass in guinea pigs, as assessed by micro-CT
and histology. Eur J Oral Sci 3:232–242
123. Suetens P (2002) Fundamentals of medical imaging. Cambridge
University Press, Cambridge
124. Stoppie N, van der Waerden JP, Jansen JA, Duyck J, Wevers M,
Naert IE (2005) Validation of microfocus computed tomography
in the evaluation of bone implant specimens. Clin Implant Dent
Relat Res 7(2):87–94
125. Cooper DM, Matyas JR, Katzenberg MA, Hallgrimsson B (2004)
Comparison of microcomputed tomographic and microradio-
graphic measurements of cortical bone porosity. Calcif Tissue
Int 74(5):437–447
126. Chappard D, Retailleau-Gaborit N, Legrand E, Basle MF, Audran
M (2005) Comparison insight bone measurements by histo-
morphometry and microCT. J Bone Miner Res 20(7):1177–1184
127. von Wilmowsky C, Schwarz S, Kerl JM, Srour S, Lell M,
Felszeghy E et al (2010) Reconstruction of a mandibular defect
with autogenous, autoclaved bone grafts and tissue engineering:
an in vivo pilot study. J Biomed Mater Res A 93(4):1510–1518
128. Boivin G, Meunier PJ (2002) The degree of mineralization of
bone tissue measured by computerized quantitative contact mi-
croradiography. Calcif Tissue Int 70(6):503–511
129. Hobson RS (1998) A pilot study of mineralization distribution in
the cortical bone of the human mandible. Arch Oral Biol
43(8):633–639
130. Engstrom A, Engfeldt B (1953) Lamellar structure of osteons
demonstrated by microradiography. Experientia 9(1):19
131. Schortinghuis J, Ruben JL, Meijer HJ, Bronckers AL, Raghoebar
GM, Stegenga B (2003) Microradiography to evaluate bone
growth into a rat mandibular defect. Arch Oral Biol
48(2):155–160
372
132. Donath K, Breuner G (1982) A method for the study of undecal-
cified bones and teeth with attached soft tissues. The Sage–Schliff
(sawing and grinding) technique. J Oral Pathol 11(4):318–326
133. Grahma J (1955) A technique of microradiography. Radiography
246:120–127
134. Hahn M, Vogel M, Schultz C, Niecke M, Delling G (1992)
Histologic reactions of the bone–implant zone and cortical bone
area after long-term hip replacement. Chirurg 63(11):958–963
135. Stanford CM, Jacobson PA, Eanes ED, Lembke LA, Midura RJ
(1995) Rapidly forming apatitic mineral in an osteoblastic cell
line (UMR 106-01 BSP). J Biol Chem 270(16):9420–9428
136. Kashiwa HK, Park HZ (1976) Light microscopic localization of
labile calcium in hypertrophied chondrocytes of long bone with
alizarin red S. J Histochem Cytochem 24(5):634–642
137. Bendick PJ, Glover JL, Hankin R, Reilly MK, Dalsing MC,
Waller BF (1988) Carotid plaque morphology: correlation of
duplex sonography with histology. Ann Vasc Surg 2(1):6–13
138. von der Mark K, von der Mark H (1977) The role of three genet-
ically distinct collagen types in endochondral ossification and cal-
cification of cartilage. J Bone Joint Surg Br 59-B(4):458–464
139. Irie K, Zalzal S, Ozawa H, McKee MD, Nanci A (1998) Mor-
phological and immunocytochemical characterization of primary
osteogenic cell cultures derived from fetal rat cranial tissue. Anat
Rec 252(4):554–567
140. Malaval L, Modrowski D, Gupta AK, Aubin JE (1994) Cel-
lular expression of bone-related proteins during in vitro oste-
ogenesis in rat bone marrow stromal cell cultures. J Cell
Physiol 158(3):555–572
141. Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L,
Tassinari MS et al (1990) Progressive development of the rat
osteoblast phenotype in vitro: reciprocal relationships in expres-
sion of genes associated with osteoblast proliferation and differ-
entiation during formation of the bone extracellular matrix. J Cell
Physiol 143(3):420–430
142. Charles P, Mosekilde L, Risteli L, Risteli J, Eriksen EF (1994)
Assessment of bone remodeling using biochemical indicators of
type I collagen synthesis and degradation: relation to calcium
kinetics. Bone Miner 24(2):81–94
143. Christenson RH (1997) Biochemical markers of bone metabo-
lism: an overview. Clin Biochem 30(8):573–593
Oral Maxillofac Surg (2014) 18:355–372
144. Bhatnagar RS, Qian JJ, Wedrychowska A, Sadeghi M, Wu YM,
Smith N (1999) Design of biomimetic habitats for tissue engi-
neering with P-15, a synthetic peptide analogue of collagen.
Tissue Eng 5(1):53–65
145. Nguyen H, Qian JJ, Bhatnagar RS, Li S (2003) Enhanced cell
attachment and osteoblastic activity by P-15 peptide-coated
matrix in hydrogels. Biochem Biophys Res Commun
311(1):179–186
146. Swaminathan R (2001) Biochemical markers of bone turnover.
Clin Chim Acta 313(1–2):95–105
147. Price CP, Thompson PW (1995) The role of biochemical tests in
the screening and monitoring of osteoporosis. Ann Clin Biochem
32(Pt 3):244–260
148. Thorwarth M, Rupprecht S, Falk S, Felszeghy E, Wiltfang J,
Schlegel KA (2005) Expression of bone matrix proteins during
de novo bone formation using a bovine collagen and platelet-rich
plasma (prp)—an immunohistochemical analysis. Biomaterials
26(15):2575–2584
149. Thorwarth M, Wehrhan F, Schultze-Mosgau S, Wiltfang J,
Schlegel KA (2006) PRP modulates expression of bone matrix
proteins in vivo without long-term effects on bone formation.
Bone 38(1):30–40
150. Frost HM, Villanueva AR, Roth H, Stanisavljevic S (1961) Tet-
racycline bone labeling. J New Drugs 1:206–216
151. Jee WS (2005) The past, present, and future of bone morphom-
etry: its contribution to an improved understanding of bone biol-
ogy. J Bone Miner Metab 23(Suppl):1–10
152. Regauer M, Jurgens I, Kotsianos D, Stutzle H, Mutschler W,
Schieker M (2005) New-bone formation by osteogenic protein-
1 and autogenic bone marrow in a critical tibial defect model in
sheep. Zentralbl Chir 130(4):338–345
153. Knöfler W, Graf H, Gröschel T, Löwicke G (1990) Bone reaction
caused by biomaterials. Z Zahnärztliche Implantol 6:145–152
154. Donath K (1988) The ‘sawing and grinding’ technique for the
preparation of histologic samples of non-cutable tissues and
materials. Der Präparator 34:197–206
155. Sverzut AT, Crippa GE, Morra M, de Oliveira PT, Beloti MM,
Rosa AL (2012) Effects of type I collagen coating on titanium
osseointegration: histomorphometric, cellular and molecular anal-
yses. Biomed Mater 7(3):035007
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