Invited Review

M. Retzepi The effect of diabetes mellitus on

N. Donos
osseous healing

Authors’ affiliations: Key words: bone healing, diabetes mellitus, hyperglycaemia, insulin, ossification
M. Retzepi, N. Donos, Periodontology Unit,
Clinical Research Division, UCL Eastman Dental
Institute, London, UK Abstract: Diabetes mellitus and, in particular, type 1 diabetes has been associated with
impaired osseous wound healing properties. The scope of the present review is to discuss
Corresponding author:
Dr Maria Retzepi
the clinical evidence supporting a higher rate of complications during fracture healing in
Periodontology Unit diabetic patients and the histological evidence indicating impaired potential for
Clinical Research Division intramembranous and endochondral ossification in the presence of uncontrolled
UCL Eastman Dental Institute
256 Gray’s Inn Road experimental diabetes. The article further provides a synthesis of our current understanding
London of the plausible molecular mechanisms underlying the diabetic bone healing
WC1X 8LD, UK
Tel.: þ 44 207915 1135 pathophysiology and of the role of insulin treatment in promoting osseous healing in the
Fax: þ 44 207915 1137 diabetic status.
e-mail: m.retzepi@eastman.ucl.ac.uk

Diabetes mellitus is defined as a ‘‘group of or ‘‘diabetic osteopathy’’ (Bouillon 1991).

metabolic diseases characterised by hyper- The diabetic skeletal phenotype presents
glycaemia resulting from defects in insulin the following features:
secretion, insulin action or both’’ (The
Expert Committee on the Diagnosis and  diminished linear bone growth during
Classification of Diabetes Mellitus 1997). the pubertal growth spurt in adoles-
The vast majority of diabetic cases fall in cents with diabetes (Salerno et al.
one of the following categories: 1997);
 reduction of bone mineral density and
(a) Type 1 diabetes mellitus (T1DM), a
increased risk for occurrence of osteo-
condition characterised by absolute
penia and osteoporosis (Vestergaard
deficiency of insulin secretion, result-
2007);
ing from autoimmune destruction of
 increased fracture risk (Janghorbani
the insulin producing b cells of the
et al. 2007);
pancreas;
 poor osseous healing characteristics and
(b) Type 2 diabetes mellitus (T2DM), a
impaired bone regeneration potential
condition characterised by impaired
(Cozen 1972).
insulin function, i.e. resistance to in-
sulin action, combined with inade-
quate compensatory insulin secretion
response. The scope of this narrative review is to
Date:
Accepted 28 December 2009
present and synthesise the evidence emer-
Diabetes mellitus has been associated ging from preclinical studies, clinical trials
To cite this article:
Retzepi M, Donos N. The effect of diabetes mellitus on with the occurrence of a series of complica- and systematic reviews on the effect of
osseous healing. tions involving the skeletal system, collec- diabetes mellitus on the osseous healing
Clin. Oral Impl. Res. 21, 2010; 673–681.
doi: 10.1111/j.1600-0501.2010.01923.x tively referred to as ‘‘diabetic bone disease’’ process.

c 2010 John Wiley & Sons A/S

 673
Retzepi & Donos  The effect of diabetes mellitus on osseous healing
Diabetes effect on skeletal
tissue metabolism
A recent meta-analysis has demonstrated
that T1DM is clearly associated with de-
creased bone density and with significantly
increased fracture risk, whereas, on the
contrary, although the relative risk for hip
fracture is moderately increased in T2DM
patients, the bone mineral density in the
hip and the spine presents increased (Ves-
tergaard 2007). Because hyperinsulinaemia
is associated with increased bone mineral
density, it has been suggested that the
observed differential clinical presentation
may be accounted for by the discrepancy
in systemic insulin concentration between
the T1DM and the T2DM condition, i.e.
insulinopenia vs. hyperinsulinaemia, re-
spectively (Thrailkill et al. 2005).
It has been postulated that the decreased
bone mineral density in T1DM patients is
aetiopathogenetically related to a sup-
pressed bone turnover profile, which is
determined by the relative contributions
of bone formation by osteoblasts and bone
resorption by osteoclasts (McCabe 2007).
T1DM has been associated with decreased
osteoblastic activity further to several stu-
dies reporting reduced systemic levels of
osteocalcin and insulin growth factor I
(IGF-I) in T1DM patients (Pedrazzoni
et al. 1989; Olmos et al. 1994; Kemink
et al. 2000) and in in vivo models of
experimental diabetes (Shires et al. 1981;
Verhaeghe et al. 1990; Horcajada-Molteni
et al. 2001; Botolin et al. 2005). In addi-
tion, studies based on bone histomorpho-
metric indices in untreated experimental
diabetes have consistently reported dimin-
ished osteoid formation, combined with
defective bone mineral apposition rate
(Goodman & Hori 1984; Verhaeghe et al.
1990). The observations of suppressed bone
formation and osteoblast recruitment and
activity in insulin-deficient models have
been further confirmed in bone biopsy
specimens from T1DM patients (Krakauer
et al. 1995). Contrary to bone formation,
markers of bone resorption display a non-
uniform systemic expression pattern in
T1DM patients, as they may be increased
(Mathiassen et al. 1990; Olmos et al.
1994), unaltered (Kemink et al. 2000) or
even decreased (Cloos et al. 1998). In
animal models of untreated experimental
diabetes, the concentrations of bone resorp-
674 | Clin. Oral Impl. Res. 21, 2010 / 673–681
tion indices present unaltered or decreased
(Shires et al. 1981; Botolin et al. 2005; Hie
et al. 2007). In accordance with these
observations, a series of studies have sug-
gested normal or reduced osteoclastic ac-
tivity based on bone histomorphometry
indices, including osteoclast number, ero-
sion depth and erosion surface (Shires et al.
1981; Verhaeghe et al. 1990; Hou et al.
1993).
Taking into consideration that, in the
context of the bone remodelling process,
bidirectional signalling activity occurs be-
tween osteoblasts and osteoclasts, these
findings overall indicate that suppressed
osteoblastic activity rather than increased
osteoclastic activity accounts for the ob-
served osteoporotic changes characterising
the T1-diabetic bone phenotype (McCabe
2007).
Impaired microarchitectural
quality of diabetic bone
Further to the reduction in bone mineral
density, detrimental changes in the bone
morphology, mineralisation and matrix
composition contribute to the observed
deterioration in the biomechanical proper-
ties of the diabetic bone, i.e. energy absorp-
tion, torsional strength, angular deformity
(Dixit & Ekstrom 1980; Einhorn et al.
1988). With regard to diabetic bone micro-
architectural morphology, poor trabecular
connectivity, increased porosity and lower
bone spicule/marrow space ratio have been
demonstrated via quantitative bone histo-
morphometry in models of experimentally
induced diabetes (Hou et al. 1993; Suzuki
et al. 2003). Deficits in the mineralised
surface area may include decreased hydro-
xyapatite crystal perfection, lower calcium-
to-phosphate composition of the ash, as
well as reduced ash content (Einhorn
et al. 1988). Furthermore, the collagenous
matrix content is reduced (Verhaeghe et al.
1994). Spanheimer et al. (1988) reported a
48% reduction in collagen production by
bone, within 2 weeks following induction
of experimental diabetes, which could not
be accounted for merely by undernutrition
(Umpierrez et al. 1989). The collagenous
matrix also features increased glycation-
induced cross-linking, which may further
deteriorate the diabetic bone quality (Saito
et al. 2006).
Diabetes effect on osseous
healing: clinical studies
Clinical studies have demonstrated that
diabetic patients present significantly de-
layed fracture healing, as the fracture callus
is slow to appear and mature (Cozen 1972;
Loder 1988; Bibbo et al. 2001). Specifi-
cally, Loder (1988) reported that the frac-
ture union time is prolonged by 87% in
non-displaced fractures in non-neuropathic
diabetic patients compared with non-
diabetic patients. Furthermore, a retrospec-
tive study addressing the treatment success
of calcaneus fractures, reported that the
presence of diabetes was associated with a
relative risk of 3.4 for wound complications
(Folk et al. 1999). Three case series
have reported increased incidence of infec-
tious complications, delayed union, non-
union, redislocation and pseudarthrosis
following elective arthrodesis in diabetic
patients (Stuart & Morrey 1990; Papa
et al. 1993; Tisdel et al. 1995). It should
be noted however that, up to date, there is a
lack of well-designed, large-scale, epide-
miological studies reporting on the inci-
dence of complications during the osseous
healing process in the population of dia-
betic patients.
Effect of experimental diabetes
on intramembranous
ossification
Deficient intramembranous bone forma-
tion in experimental diabetes has been
histologically and histomorphometrically
confirmed in various osteotomy and frac-
ture models.
Santana et al. (2003) reported suppressed
intramembranous bone formation, using a
model of calvarial defects of 1, 1.6 and
2.1 mm in diameter, in streptozotocin-
induced experimental diabetes in mice.
Qualitative histological observations indi-
cated less developed marrow spaces and
suppressed cellularity and vascularisation
after 14 days of healing, whereas plani-
metric measurements demonstrated that
calvarial bone bridging was inhibited by
40% in diabetic animals, irrespectively
of the defect size. Follak et al. (2004a)
evaluated the effect of spontaneous experi-
mental diabetes on osseous healing of cra-
niotomy defects of various sizes in rats.
c 2010 John Wiley & Sons A/S


The authors did not detect differences in
the bone healing process of small bone
defects (0.4 mm) between poorly controlled
diabetic and healthy animals. In 0.8 mm
defects, significantly decreased trabecular
bone volume was observed in poorly
controlled diabetes at 24 and 42 days
of healing. In 1.2 mm and larger defects
however, the poorly controlled diabetic
status correlated with severe mineralisa-
tion disorders during the first 2 weeks of
healing, as indicated by suppressed
expression of dynamic histomorphometric
parameters of mineralisation, i.e. apposi-
tion, formation and timing of mineralisa-
tion. Interestingly, in this study, the
osteotomy size was an independent vari-
able in predicting successful osseous
repair, irrespective of the diabetic meta-
bolic status.
The periosteal callus represents an addi-
tional site featuring intramembranous ossi-
fication during the fracture healing process.
Histological reports on the effect of sponta-
neous experimental diabetes on periosteal
callus formation in femoral fracture mod-
els, indicated diminished osteoid deposi-
tion at 2 and 4 days of healing and more
immature appearance of the newly formed
woven bone 7 days after the injury (Beam
et al. 2002; Tyndall et al. 2003).
Lu et al. (2003) have employed the
marrow ablation model in order to study
the effect of streptozotocin-induced dia-
betes on intramembranous bone formation.
The model involved removal of the trabe-
cular bone and marrow from the marrow
cavity of the diaphyseal portion of the tibia,
which was followed by a synchronised
series of intramembranous bone repair
events during a period of 10 days. Histo-
morphometric evaluation indicated similar
amounts of mesenchymal tissue on day 4
in diabetic and healthy animals, whereas
decreased bone and marrow formation were
observed after 6 and 10 days of healing in
the diabetic group.
The distraction osteogenesis model has
also been used in order to study the effect of
experimental diabetes on intramembra-
nous bone regeneration potential. Fowlkes
et al. (2008) reported that after 15 days of
healing, bone bridging was 19.3  12.5%
in the diabetic animals vs. 52.4  6.2% in
the control group.
Summarising the available histological
evidence, experimental diabetes has been
c 2010 John Wiley & Sons A/S
 675 |
Retzepi & Donos  The effect of diabetes mellitus on osseous healing
associated with impaired intramembranous
bone formation potential, mostly after the
first and during the second week of osseous
healing. It is noteworthy that the type and
size of the osseous defect seem to consti-
tute independent determinants of the ac-
tual impact of diabetes on the ossification
process.
Effect of experimental diabetes
on endochondral ossification
Qualitative histological studies on the ef-
fect of experimental diabetes on the frac-
ture gap callus, a site where endochondral
ossification occurs, demonstrated reduced
amounts of undifferentiated mesenchymal
cells (MSCs) at 4 days of healing (Beam
et al. 2002; Tyndall et al. 2003). Further-
more, at 4 and 7 days after the injury, the
diabetic gap callus featured reduced carti-
lage formation with less proliferating chon-
drocytes (Beam et al. 2002; Tyndall et al.
2003; Gandhi et al. 2005). Histological
observations in a similar fracture model,
at later healing periods, correlated the dia-
betic status with delayed differentiation of
the callus at 2 weeks of healing, whereas,
at 4 weeks, prominent hypertrophic chon-
drocytes undergoing ossification were still
present, thus indicating a delay in cellular
differentiation (Follak et al. 2005).
Histomorphometric evaluation of the
diabetic gap callus at 2 and 4 weeks of
healing, demonstrated significantly re-
duced dynamic parameters of mineralisa-
tion, including apposition, formation and
timing of mineralisation (Follak et al.
2005). These observations are in line with
a previous report by the same group, which
evaluated histomorphometrically the effect
of spontaneous diabetes on the osseous
healing process, using a model of 10 mm
circular osteotomies created in the distal
femur (Follak et al. 2004b). The authors
reported that the poorly controlled diabetic
state correlated with suppressed and re-
tarded mineralisation process, evidenced
by significantly reduced apposition, forma-
tion and timing of the mineralisation up to
the 14th postoperative day. Histomorpho-
metric evaluation of the diabetic gap callus
at late healing periods of 6 and 8 weeks,
indicated incomplete bridging of the cor-
tice/fracture callus, diminished remodel-
ling activity and decreased percent
mineralised area in the diabetic animals
compared with healthy controls (Gandhi
et al. 2005).
The histological and histomorphometri-
cal observations are in line with evidence of
reduced biomechanical properties of the
fracture callus in uncontrolled experimen-
tal diabetes (Macey et al. 1989). The
diabetic femoral fracture callus presents
delayed recovery of its structural properties
and material strength, and more specifi-
cally reduced shear stress, torque to failure
and stiffness at 2, 4, 6 and 8 weeks
of healing (Macey et al. 1989; Funk
et al. 2000; Beam et al. 2002; Follak et al.
2005).
In conclusion, experimental diabetes is
associated with reduced cellularity in
the fracture milieu during the first post-
traumatic week and with subsequently
delayed and impaired cartilage differentia-
tion and mineralisation, which account
for impaired histological and biomechani-
cal healing parameters observed at the
late stages of the endochondral ossification
process.
Effect of diabetes on
osseointegration
Clinical studies
Evidence based on clinical trials addressing
the effect of diabetes on the survival rates of
titanium implants is equivocal at present.
Among the controlled clinical studies
available, some report an increased risk
for implant failure in diabetic patients
compared with healthy controls with a
relative risk of 2.75 (95% confidence inter-
val: 1.46–5.18) (Morris et al. 2000; Moy
et al. 2005), whereas others do not support
an association between diabetes and im-
plant failure (Smith et al. 1992). Most of
the case-series studies on implant survival
in diabetic patients support a trend of
increased early vs. late implant failure
(Mombelli & Cionca 2006), although there
have also been reports of increased failure
rate after functional loading, presumably
associated to impaired bone remodelling
potential (Shernoff et al. 1994; Fiorellini
et al. 2000). Furthermore, one study re-
ported higher crestal bone loss during the
first year of implant loading in T2DM
patients (Accursi 2000). Taking into
consideration that the vast majority of
Clin. Oral Impl. Res. 21, 2010 / 673–681
Retzepi & Donos  The effect of diabetes mellitus on osseous healing
participants in these clinical trials were
well-controlled T2DM patients, it may be
concluded that implant placement is not
contraindicated in diabetic patients pre-
senting good metabolic control (Mombelli
& Cionca 2006).
Preclinical studies
Evidence of delayed peri-implant bone for-
mation in uncontrolled experimental dia-
betes is based on qualitative observations of
more immature histological appearance of
the newly formed bone following place-
ment of titanium implants in the femur
(Nevins et al. 1998) or tibia (Giglio et al.
2000) of diabetic rats. These results were
in accordance with previous reports of im-
paired new bone formation following place-
ment of hydroxyapatite-coated implants
in the tibiae of diabetic rats (Iyama et al.
1997). In accordance with the qualitative
histological observations, significantly
reduced bone-to-implant contact has been
reported in diabetic animals after 2, 3, 4, 8
and 12 weeks of healing following
placement of titanium implants (Nevins
et al. 1998; Siqueira et al. 2003; Ottoni &
Chopard 2004), although the figures
were comparable to healthy controls at 12
postoperative weeks in two other studies
(Gerritsen et al. 2000; Retzepi et al. 2010).
The observed discrepancy may be attribu-
ted to different models of endosseous
implantation, i.e. endochondral vs. intra-
membranous bones and/or to differences
in the implant surfaces employed, i.e.
smooth vs. rough. Furthermore, a reduc-
tion in peri-implant bone density (Gerrit-
sen et al. 2000) and up to 50% reduction in
the amount of newly formed bone after the
first 2 weeks of healing (Takeshita et al.
1998; Giglio et al. 2000; McCracken et al.
2000; Siqueira et al. 2003; Ottoni & Cho-
pard 2004) have also been reported. With
regard to the effect of experimental diabetes
on the biomechanical properties of endoss-
eous implants, reduced removal torque
values have been observed at 8 and 12
weeks postimplantation (Margonar et al.
2003).
It may be therefore concluded that,
although osseointegration is feasible in
uncontrolled experimental diabetes, this
condition is associated with impaired
peri-implant bone formation, the aetio-
pathologic features of which remain largely
unknown.
676 | Clin. Oral Impl. Res. 21, 2010 / 673–681
Molecular mechanisms
implicated in impaired diabetic
bone healing
The impaired osteoid matrix production
and the reduced cellularity observed during
the early osseous healing period in uncon-
trolled experimental diabetes may stem
from deficits in the recruitment of MSCs
in the osteoblastic lineage, impaired differ-
entiation and proliferation of cells of the
osteoblastic lineage and/or suppressed
osteoblastic cell activity (Weiss & Reddi
1980; McCabe 2007).
The hypothesis of impaired commit-
ment of MSCs in the osteoblastic lineage
during osseous healing in uncontrolled dia-
betes is supported by evidence of sup-
pressed expression of transcription factors
required for the acquisition of the osteo-
blastic phenotype. Specifically, suppressed
mRNA expression of the Dlx5 and Runx-2
transcription factors, which control acqui-
sition of the osteoblastic phenotype, has
been observed at 4 and 6 days of intramem-
branous bone healing in the marrow abla-
tion model (Lu et al. 2003). These data
indicate that the diminished bone forma-
tion observed in the diabetic status could be
related to deficiencies in the differentiation
of MSCs to osteoprogenitor cells and in
the osteoblastic cell maturation (McCabe
2007).
Reduced proliferation of osteoblastic
cells has been also suggested as a plausible
mechanism for impaired bone healing.
This is supported by studies associating
the decreased cellularity commonly ob-
served in the diabetic callus, with an up
to 50% reduction in immunohistochem-
ical indices of the cell proliferation rate in
the periosteal and in the gap callus at 2, 4
and 7 days of healing (Beam et al. 2002;
Gebauer et al. 2002; Tyndall et al. 2003;
Gandhi et al. 2005). In accordance with
these immunohistochemical observations,
gene expression profiling investigation has
indicated that uncontrolled diabetes fea-
tured downregulation of pathways related
to cell division, energy production and
osteogenesis during the proliferative phase
of intramembranous bone healing follow-
ing guided bone regeneration application
(Retzepi 2009). The underlying causes of
the reduced cell proliferation rates remain,
however, largely unknown. It has been
hypothesised that diabetes may suppress
the levels of critical growth factors, thus
disrupting the normal progression of the
inflammatory phase and osteogenesis, dur-
ing the early osseous healing stages (Tyn-
dall et al. 2003; Gandhi et al. 2005). An
initial report by Kawaguchi et al. (1994)
indicated decreased expression of basic fi-
broblast growth factor (FGF), an established
mitogenic factor for MSCs during osseous
healing, in the diabetic fracture callus at 1
and 3 weeks following the injury. Reduced
mRNA and protein expression of platelet-
derived growth factor (PDGF), another po-
tent mitogenic factor, have also been
observed in femoral fractures of sponta-
neously diabetic rats during the first heal-
ing week and has been correlated with
reduced cell proliferation rates during the
early phases of endochondral bone forma-
tion (Tyndall et al. 2003). A recent study
by Gandhi et al. (2005) confirmed the
reduction in PDGF, transforming growth
factor b1 (TGF-b1), IGF-I and vascular
endothelial growth factor protein expres-
sion levels at femoral fracture sites at 4
and 7 days of healing, in poorly controlled
spontaneously diabetic rodents. The results
of various studies evaluating the efficacy of
local delivery of growth factors in promot-
ing osseous healing in uncontrolled dia-
betes support further the hypothesis of
deficient production and/or action of
growth factors and hormones in the oss-
eous healing process in uncontrolled dia-
betes. Thaller et al. (1995) reported that
delivery of IGF-I in critical size calvarial
defects in streptozotocin-induced diabetic
rats enhanced the osseous healing process.
Santana & Trackman (2006) reported that
exogenous application of 5 mg of rhFGF-2
promoted bone bridging by 2.6-fold in cal-
varial defects in streptozotocin-induced
diabetic mice after 14 days of healing.
The implication of reduced activity of
osteoblastic cells in the impaired bone
healing potential in the presence of diabetes
is supported by evidence of substantially
reduced mRNA expression of the specific
bone matrix protein osteocalcin and of type
1 collagen at 4 and 6 days of intramem-
branous bone healing in the marrow abla-
tion model (Lu et al. 2003). This is in
accordance with previous reports of a 50–
55% reduction in total collagen content
(Macey et al. 1989) and 54–70% decrease
in type X collagen content (Topping et al.
1994) during the first 2 weeks of fracture
c 2010 John Wiley & Sons A/S


healing in uncontrolled experimental dia-
betes.
In conclusion, suppressed differentia-
tion, proliferation and/or bone forming
capacity of osteoblastic cells during the
critical early healing period have been im-
plicated as plausible pathogenetic mechan-
isms underlying poor bone formation in
uncontrolled diabetes.
Insulin-mediated glycaemic
control reverses diabetic bone
pathophysiology
The results of clinical studies assessing the
relationship between diminished bone
mineral density and glycaemic control in
T1DM are conflicting. Among the clinical
studies supporting an impact of T1DM
on bone mineral density, several support
a correlation between decreased bone
mineral density and poor metabolic control
(Lettgen et al. 1995; Valerio et al. 2002;
Heap et al. 2004), whereas others did not
show an association (Gunczler et al. 1998).
Various experimental studies have de-
monstrated that insulin treatment exerts a
preventive effect on the pathophysiology of
osseous tissue observed in streptozotocin-
induced experimental diabetes (Hough
et al. 1981; Hou et al. 1991; Suzuki et al.
2003) and in spontaneous experimental
diabetes (Verhaeghe et al. 1997). The tim-
ing of insulin treatment is considered cri-
tical. Early initiation of insulin therapy in
experimental diabetes can prevent the ex-
pression of the diabetic skeletal phenotype,
whereas, in later stages, insulin delivery
can only partly normalise parameters char-
acterising the diabetic bone pathology
(Hough et al. 1981). It has been suggested
that insulin normalises diabetic bone me-
tabolism by enhancing bone formation
and/or by preventing bone resorption (Dixit
& Ekstrom 1980; Haffner & Bauer 1993;
Krakauer et al. 1995).
Deficient osseous healing in experimen-
tal diabetes is also reversible via insulin
treatment, because a series of in vivo stu-
dies indicate that the histomorphometric,
biomechanical and biochemical indices
during osseous healing in experimentally
diabetic animals with insulin-mediated,
good metabolic control are comparable
to healthy controls (Goodman & Hori
1984; Hou et al. 1993). Specifically, it
c 2010 John Wiley & Sons A/S
 677 |
Retzepi & Donos  The effect of diabetes mellitus on osseous healing
has been demonstrated that systemic in-
sulin treatment improves the biomechani-
cal parameters in streptozotocin-induced
experimental diabetes during the late stages
of fracture healing (Herbsman et al. 1968;
Macey et al. 1989). Furthermore, Beam
et al. (2002) showed that insulin therapy
normalised the biomechanical parameters,
i.e. percent mechanical stiffness, torque to
failure and ultimate shear stress in sponta-
neously diabetic animals. In accordance
with these observations, good metabolic
control achieved via systemic insulin de-
livery in spontaneously diabetic rats nor-
malised the histomorphometric indices of
mineralisation and callus bone content,
during later stages of fracture healing, com-
pared with diabetic animals with low me-
tabolic control (Beam et al. 2002). These
reports are in accordance with evidence of
improved bone bridging of osteotomy cal-
varial (Santana et al. 2003) and femoral
(Follak et al. 2004b) defects in well-
controlled diabetic animals via insulin
treatment. Insulin-mediated metabolic
control has been also shown to improve
the histomorphometric parameters of
osseointegration of titanium implants in
experimental diabetes (Fiorellini et al.
1999; Siqueira et al. 2003).
Qualitative histological observations
during the early stages of osseous healing,
indicated similar intramembranous and en-
dochondral bone formation potential in the
periosteal and gap callus in well-controlled
insulin-treated diabetic animals compared
with non-diabetic controls at 2, 4 and 7
days of fracture healing. The authors sug-
gested that these findings support the view
that insulin plays a critical role during the
early stages of fracture healing (Beam et al.
2002). Follak et al. (2005) further provided
detailed histometrical data indicating
that, in spontaneously diabetic animals,
insulin-mediated strict glycaemic control
normalised all dynamic parameters of
mineralisation, apposition, osteoid forma-
tion and timing of mineralisation compared
with the poorly compensated diabetic ani-
mals, during the first 4 weeks of femoral
fracture healing. Beam et al. (2002) inves-
tigated the impact of insulin therapy on the
cellular proliferation rates within the frac-
ture callus in experimental diabetes. They
showed that, during the first week of
fracture healing, the cellular proliferation
values in the fracture callus of insulin-
treated diabetic rats with well-compen-
sated metabolic status, were similar to
non-diabetic controls and by 25% higher
compared with poorly controlled diabetic
animals. These results are in accordance
with a previous study reporting a 27%
decrease in cell proliferation in a diabetic
explant model, which was normalised fol-
lowing insulin delivery (Weiss & Reddi
1980). A more recent study confirmed
these observations, by reporting enhanced
cellular proliferation in well-controlled vs.
poorly controlled diabetic animals, during
the first 4 weeks of healing in a femoral
fracture model (Follak et al. 2005).
Plausible pathogenetic links
between diabetes and impaired
osseous healing
Role of hyperglycaemia
In vitro studies have reported that exposure
of osteoblastic cells to increased glucose
concentrations reduces their differentiation
potential, leads to irregular bone mineral
nodule formation (Balint et al. 2001) and
suppresses cell-growth rates (Terada et al.
1998). Suppressed Runx2 mRNA levels
have been observed following short-term
exposure to hyperglycaemic conditions,
although not consistently observed in longer
term studies (Botolin et al. 2005; Botolin &
McCabe 2007). In addition, prolonged expo-
sure of osteoblastic cells to hyperglycaemic
conditions is correlated with altered gene
expression, characterised by decreased osteo-
calcin, MMP-13, collagen-1 and c-jun
mRNA levels (Zayzafoon et al. 2002; Boto-
lin & McCabe 2006). Various potential
pathogenetic mechanisms may account for
the responses of osteoblastic cells to pro-
longed hyperglycaemia, including modula-
tion of the redox state, increased activity of
the polyol-pathway, activation of the protein
kinase C pathway, and non-enzymatic gly-
cosylation of key proteins, e.g. collagen type
I and IGF-1 (McCabe 2007).
The accumulation of advanced glycation
end-products (AGEs) in the osseous tissue
as a result of non-enzymatic glycosylation
has been implicated in the pathogenesis of
diminished bone formation, in a fracture-
healing model in experimental diabetes
(Santana et al. 2003). AGEs may directly
modulate the growth, differentiation and
activity of osteoblastic cells and these effects
Clin. Oral Impl. Res. 21, 2010 / 673–681
Retzepi & Donos  The effect of diabetes mellitus on osseous healing
are possibly mediated via their interaction
with specific osteoblastic receptors (RAGEs)
(McCarthy et al. 2001; Santana et al. 2003;
Schwartz 2003; Kume et al. 2005). One
possible pathway is that AGE–RAGE inter-
actions may result in generation of reactive
oxygen intermediates and/or nuclear factor
k B (NFkB) activation in osteoblastic cells
(Lalla et al. 1998; Kislinger et al. 1999;
McCarthy et al. 2001). Another plausible
mechanism is that AGEs modification of
type-I collagen impairs integrin-mediated
adhesion of osteoblasts on the extracellular
matrix and therefore compromises their
normal bone-forming activity (McCarthy
et al. 2001; 2004).
Hypercalciuria, neuropathy and angiopathy
Hypercalciuria resulting from increased
glycosuria has been proposed as a mechan-
ism for diabetic osteopathy, although the
results are not consistent among different
studies and the effect cannot be accounted
for by the magnitude of the changes ob-
served (Carnevale et al. 2004). Altered
systemic excretion profile of calcium-
regulating hormones, such as parathormon
and 25-hydroxyvitamin D has been further
suggested to play a role in diabetic bone
phenotype (Alrefai et al. 2002), although
this mechanism may not account for the
regional pattern of the changes observed
(Ahmad et al. 2004).
Neuropathy and angiopathy have been
suggested as the main features behind local
alterations of diabetic bone metabolism.
Peripheral neuropathy is considered as an
independent risk factor for bone mineral
density reduction among type 1 diabetics
(Piepkorn et al. 1997; Rix et al. 1999), as it
may exert direct effects on bone through
neurotransmitter action on bone cells
(Bjurholm et al. 1992; Lerner 2002). Alter-
natively, diabetic osteopenia may result
from disturbed vasoregulation caused by
neuropathy (Rajbhandari et al. 2002).
Although increased bone resorption has
been observed in the presence of abnormal
blood flow in an experimental T2DM
model (Laroche 2002), the osteoporotic
changes preceded the onset of microvascu-
lar changes, thus indicating that microan-
giopathy did not play a major role in the
pathogenesis of osteopenia (Amir et al.
2002). In conclusion, the currently avail-
able evidence seems to support that the
main mechanisms underlying diabetic
678 | Clin. Oral Impl. Res. 21, 2010 / 673–681
bone pathophysiology are hyperglycaemia
and/or hypoinsulinaemia (McCabe 2007).
Hypoinsulinaemia
Insulin appears to exert a direct anabolic
effect on the bone formation process during
osseous healing in vivo (Cornish et al.
1996). In vitro studies have indicated that
insulin exerts direct anabolic effects on
osteoblastic cells, evidenced by increased
proliferation rates (Hashizume & Yamagu-
chi 1993), collagen synthesis (Craig et al.
1989), alkaline phosphatase production
(Canalis 1983) and glucose uptake (Hahn
et al. 1988; Ituarte et al. 1989). Possible
direct actions of insulin on bone cells may
include mitogenic stimulation of bone-
forming cells, coupled with inhibition of
apoptosis (Thrailkill et al. 2005). In addi-
tion to its direct effects on cells of the
osseous tissue, insulin may also synergis-
tically enhance the response of bone cells
to other anabolic growth factors for osseous
tissue, such as IGF-I and PTH (Conover
et al. 1996; Suzuki et al. 2003). It may be
therefore suggested that, because insulin
constitutes a well-documented anabolic
factor for osseous tissue, T1DM-associated
insulinopenia may, at least partly, account
for the observed bone loss and impaired
osseous healing potential.
Although it has been documented that
abnormal bone repair in experimental dia-
betes is insulin dependent because the
deficient osseous healing process is re-
versed by insulin treatment, it is at present
unclear whether this effect is primarily due
to the insulin deficiency or to the hyper-
glycaemia characterising the diabetic status
(Thrailkill et al. 2005). It has been advo-
cated that the relative contribution of in-
sulinopenia may be more significant than
moderate hyperglycaemia, as normalisa-
tion of diabetic fracture parameters can be
achieved with insulin treatment, despite
suboptimal metabolic control (Gooch
et al. 2000). Gandhi et al. (2005) evaluated
the direct effects of local insulin delivery on
diabetic bone repair, in the absence of
systemic metabolic alterations. Intrame-
dullary placed pellets of sustained insulin
release at femoral fracture sites in sponta-
neously diabetic rats normalised cellular
proliferation in the periosteal and gap
callus, as well as chondrogenesis, within
the early diabetic fracture callus. During
later healing stages, insulin treatment
further increased the callus content and
mineralisation, significantly improving the
fracture gap bridging. The authors suggested
that insulin plays a critical role in directly
promoting the fracture healing potential and
that impaired diabetic osseous healing may
be associated with reduced local insulin
levels, as a result of decreased systemic
insulin levels in the diabetic state.
Conclusions
Diabetes mellitus has been consistently
associated with deficient metabolism of
the skeletal tissue, which, especially in
T1DM, is primarily related to suppressed
osteoblastic activity and reduced bone for-
mation potential, irrespectively of the type
of bone, the location and the mechanical
loading characteristics. Clinical and in vivo
studies have further established that im-
paired intramembranous and endochondral
ossification constitute dominant pathophy-
siological traits characterising diabetic bone
disease. Nonetheless, well-designed, epide-
miological studies reporting on the preva-
lence, incidence and type of osseous
healing complications in large populations
of diabetic patients are warranted. Sys-
temic insulin treatment can promote the
intramembranous and endochondral oss-
eous healing potential in diabetes, which
may be associated with its direct mito-
genic, stimulatory and anti-apoptotic
effects on osteoblasts and with its synergis-
tic effect with other growth factors.
Several plausible molecular mechanisms
have been implicated in the impaired dia-
betic bone healing and are mainly related to
suppressed differentiation, proliferation
and/or bone forming capacity of osteoblas-
tic cells during the critical early phases of
the osseous healing process. The currently
available evidence seems to support that
the main mechanisms underlying diabetic
bone pathophysiology are hyperglycaemia
and/or hypoinsulinaemia in the case of
T1DM. Future studies should be directed
at dissecting the roles of hyperglycaemia
and hypoinsulinaemia on the pathogenesis
of the impaired bone healing in the diabetic
status and at implementing the acquired
knowledge for the design of novel tissue
engineering and drug delivery therapeutic
strategies for the reconstruction of osseous
defects in diabetic patients.
c 2010 John Wiley & Sons A/S


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