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Sheldon S. Lin, MD, is an assistant professor in
the Department of Orthopaedics at UMDNJ-New Jersey
Medical School (NJMS). |
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Impaired Bone
Healing in Patients with Diabetes Mellitus
by Sheldon
S. Lin, MD
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this article as an Adobe PDF (204KB)
Fracture healing is a process of restoring the structural
and biological properties of injured bone. It has been
well documented that diabetes mellitus (DM), a systemic
disease affecting 17 million Americans, causes increased
healing time with a concomitant increase in delayed
unions and nonunions. Unfortunately, the specific mechanism
for the delayed fracture healing in patients with diabetes
has yet to be elucidated.
The purpose of our research is to evaluate the role
of insulin and glucose control on the fracture healing
process using our recently established femur fracture
model in diabetic BB Wistar rats. We theorize that insulin
plays a critical role in the fracture callus, especially
on the early inflammatory phase and the expression of
local growth factors, and its relative absence leads
to impaired fracture healing in diabetics.
Annually, 6 million fractures are treated in the U.S.,
with 5-10% exhibiting complications such as delayed
union or non-union. Etiologies of impaired fracture
healing include smoking, open fractures, presence of
underlying infection, certain medications (i.e. steroids)
as well as systemic disease such as diabetes mellitus
(DM). It is estimated that more than 30 million Americans
will be diagnosed with diabetes within the next decade.
The effects of diabetes upon fracture healing have
been well documented with increased healing time (2-3
times the normal rate) and concomitant increased complications
(delayed union, non-union, etc). In order to study these
problems, our lab has established a diabetic femur fracture
model for analyzing the effect of tight blood glucose
and the role of early critical growth factors upon impaired
diabetic fracture healing.
Diabetic Femur Fracture
Model
Previous diabetic models were induced with cytotoxic
agents (alloxan, streptozotocin) that preferentially
destroy pancreatic beta cells. These cytotoxins result
in a clinical condition of insulin-dependent DM, Type
I diabetes. The key criticism of this method was its
inability to determine whether the deficient diabetic
fracture healing process is due to the systemic effect
of the cytotoxins, malnutrition and/or the diabetic
condition itself.
Our recently published diabetic femur fracture model
utilizes BB Wistar rats, which spontaneously develop
diabetes through the autoimmune destruction of pancreatic
â cells. The spontaneous onset of diabetes in
the BB Wistar rat confers advantages over the viral,
chemical and immunological induction of DM. Within seven
days after glycosuria, the beta cells were completely
destroyed and if untreated, marked wasting of the body
tissue, including fat, muscles, protein, dehydration
and ketosis supervene. Death usually resulted within
five to 10 days after onset. These conditions were resolved
with insulin treatment. The BB Wistar rat currently
represents a close homology of human Type I diabetes
in a laboratory animal.
Diabetic Femur Fracture
Model: Effect of Blood Glucose Control
Our published studies were able to demonstrate that
femur fracture healing in poorly controlled diabetic
rats (blood glucose > 300 mg/dl) is reproducibly
delayed compared to non-diabetic control animals. Tight
glucose control, through increased insulin treatment
resulting in blood glucose val-ues of approximately
120 mg/dl, ameliorates impaired diabetic fracture healing.
The average blood glucose values for the “loose
control” (LC) and “tight control”
(TC), and non-diabetic (non-DM) animal were 369.0, 118.9,
and 70.5 mg/dl, respectively. The non-diabetic and diabetic
(LC, TC) fracture calluses were analyzed for cellular
proliferation, histology, growth factor protein/gene
expression and biomechanical parameters to identify
differences in the fracture healing process.
Cellular Proliferation
Cellular proliferation rates were determined at two,
four and seven days post-fracture within the fracture
callus by immunohistochemical staining for bromodeoxyuridine
(BrdU). Its expression can only be detected in the nucleus
of proliferating cells and is a specific marker of proliferating
cells. We found a statistically significant reduction
in proliferating cells in the LC diabetic fracture callus
as compared to the non-diabetic callus at days two and
four post-fracture. With tight physiologic glucose control,
the number of proliferating cells in the TC diabetic
fracture callus was significantly increased compared
with the LC diabetic callus.
Qualitative Histology
Histological examination at two days post-fracture
showed a fracture callus that was similar in all three
groups. However, the LC diabetic fracture callus lacked
small areas of intramembranous bone formation found
in the non-diabetic and TC diabetic fracture callus.
At four days, there seemed to be a greater number of
pre-chondrocytes and immature, proliferating chondrocytes
in non-diabetic and the TC diabetic fracture callus
compared to the LC diabetic callus. Although the callus
area did not differ between the different groups, there
appeared to be a greater amount of newly formed osteoid
in non-diabetic and TC diabetic fracture callus compared
to the LC diabetic fracture callus. At seven days, the
periosteal callus appeared similar in all three groups
but the gap callus in non-diabetic and TC diabetic animals
appeared to be more advanced than in LC diabetic animals,
with greater areas of cartilage formation characterized
by more proliferating and hypertrophic chondrocytes.
(See figures 1, 2.)
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Figure 1: SAM images and corresponding
histology at 8 weeks: pseudo-color maps of acoustic
impedence values determined by the SAM, with corresponding
histology in A) non-diabetic and B) tightly controlled
diabetic animals. CB= cortical bone, GC=gap callus,
PC=periosteal callus. |
Biomechanical Testing
Biomechanical testing was performed using scanning
acoustic microscopy (SAM) and a servo-hydraulics mechanical
testing apparatus (MTS machine). Acoustic microscopy
is a non-destructive ultrasonic technique that has recently
been used to study bone remodeling. A positive correlation
exists for cortical bone between the acoustic impedance
and the mechanical stiffness. The acoustic impedance
values in the LC diabetic fracture callus were significantly
lower than in the non-diabetic fracture callus at six
weeks (4.79±0.34* vs. 5.62±0.50; p <
0.05) and at eight weeks (5.31±0.34 vs 6.11±0.21;
p < 0.05). Acoustic impedance values in the TC diabetic
callus were normalized. Levels at six and eight weeks
were 5.70±0.23 and 6.20±0.29. They were
not significantly lower than non-diabetic callus. There
was a significant reduction in callus bone content in
LC diabetic animals compared to both TC diabetic and
non-diabetic animals. The delay in endochondral ossification
and subsequent remodeling in the LC diabetic callus
was normalized in TC diabetic animals at six and eight
weeks.
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Figure 2: TGF-b1 Immunolocalization in
Periosteal Callus: TGF-b1localization by immunohistochemistry
at 4 days post fracture in A) non-DM and B) LC-DM
callus. C= cortical bone, OB= osteoblast, OC= osteocyte,
OS= osteoid. |
Mechanical testing supported the earlier parameters
of the positive effect of tight blood glucose control.
At six weeks post fracture, torque to failure and stiffness
in the TC diabetic fractured femur were normalized to
values not statistically different from non-diabetic
values. Values of polar moment of inertia and area were
also statistically lower in the LC diabetic fracture
callus when compared to the non-diabetic callus. These
geometric parameters were normalized in the TC diabetic
callus. When data was normalized to contralateral, intact
limbs, both percent of torque to failure and percent
of stiffness were significantly reduced in LC diabetic
animals compared to nondiabetic and TC diabetic animals.
In an attempt to understand possible mechanisms of
impaired DM fracture healing process and its resolution
with systemic insulin (tight control), we theorized
impaired DM fracture healing occurs secondary to the
decreased and/ or uncoordinated release of local growth
factors at the fracture site, an impairment of the early
inflammatory phase leading to insufficient growth factor
production. With the established model, the presence
and expression of the early critical growth factor were
analyzed using immunohistochemistry and quantitative
competitive RT-PCR.
Immunohistochemistry
Significant reduction in the local growth factor expression
and production in the LC diabetic fracture callus was
observed compared to the non-diabetic callus. Although
most of the patterns identified for the localization
of each of the growth factors investigated at 4 days
were comparable between the non-diabetic and LC diabetic
callus, there appeared to be less overall staining in
all factors in the LC diabetic callus when compared
to the non-diabetic callus. This suggested that the
delay in the diabetic fracture healing process may be
preceded by a reduction in early growth factors critical
for endochondral bone formation.
mRNA Quantitation
The amounts of PDGF-B, TGF-ß1 and IGF-I mRNA
were significantly reduced in DM animals when compared
to controls. By seven days, PDGFB and IGF-I mRNA levels
were normalized in DM animals while TGF-â1 expression
remained significantly lower in DM callus. VEGF mRNA
levels were significantly reduced in the DM when compared
to the non-DM fracture callus at day seven.
Our data supports the theory that the deficiency in
DM fracture healing occurs early, with a delay in the
expression of these growth factors concurrent with a
histologic delay in the progression of chondrogenesis.
These findings provide the impetus for our lab’s
current focus into the application of fracture healing
adjuncts including local insulin delivery, low intensity
pulsed ultrasound, pulsed electromagnetic field, and
local growth factor delivery, as possible fracture healing
treatments in diabetic patients.
Sheldon S. Lin, MD, attended Pennsylvania State
University as part of a six year BS-MD program and earned
his MD from Jefferson Medical College. He continued
his studies as research fellow under Rocky Tuan, PhD,
his orthopedic residency under Richard Rothman, MD,
PhD, at Thomas Jefferson University Hospital and his
foot and ankle fellowship at the Medical College of
Wisconsin. He has held an appointment since 1996 in
the Department of Orthopaedics of UMDNJ-New Jersey Medical
School. His research focuses on the impaired bone healing
process.
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