Download Work performed at the Midwest Orthopaedic Research Foundation

Animal Modeling of Interventions to Promote
Fracture Healing in the Face of Adverse Comorbidities
Work performed at the
Midwest Orthopaedic Research Foundation, Minneapolis, MN
1Xinqian
4David
Chen, MD; 1Joan E. Bechtold, PhD; 2Dean T. Tsukayama, MD; 3Andrew H. Schmidt, MD;
W. Polly, Jr., MD; 5Robert S. Gilley, DVM, PhD; 3Richard F. Kyle, MD; 1William D. Lew, MS
1Midwest
Orthopaedic Research Foundation, Minneapolis, MN
2Division of Infectious Diseases, Department of Medicine, Hennepin County Medical Center, Minneapolis, MN
3Orthopaedic Surgery Department, Hennepin County Medical Center, Minneapolis, MN
4Department of Orthopaedic Surgery, University of Minnesota, Minneapolis, MN
5School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA
Clinical Problem
Fracture healing is impaired when
associated with ...
Infection
Diabetes
Smoking
Glucocorticoid therapy
These comorbidities increase the risk
of complications such as ...
Delayed union or nonunion
Wound necrosis
Bony lysis
Loss of fracture fixation
Increased incidence of infection
Compromised biologic and mechanical
properties of bone
Interventions are needed that stimulate bone formation in these circumstances
Preclinical animal modeling was used to evaluate therapeutic modalities that may potentially counteract
the effects of these comorbidities
Internally-stabilized segmental defect in rat femur
Closed fracture in rat femur
Intramuscular osteoinduction model in rat
Findings from this work require further research and confirmatory clinical studies
ANIMAL MODELS
Internally-Stabilized Segmental Defect in Rat Femur
6 mm segmental defect surgically created in femur, stabilized with polyacetyl plate
and 6 Kirschner wires
Used to simulate acute contamination or chronic infection
Chronic infection model:
- Collagen carrier wetted with bacterial suspension and placed in defect
- Animal recovers for several weeks while contamination progresses to chronic infection
- Defect then surgically debrided and treated with bone morphogenetic protein (BMP) and local and systemic antibiotic while leaving implant in place
(Hollinger and Kleinschmidt. J Craniofacial Surg. 1990;1:60-8)
Closed Fracture in Rat Femur
Intramuscular Osteoinduction Model in Rat
Retrograde intramedullary pin surgically placed
in femur
Mid-diaphyseal transverse closed femur fracture
created using drop-weight impaction device
Bacteria may be introduced locally or
hematogenously; animal may be made to be
diabetic, or receive glucocorticoid therapy or
exposed to nicotine/tobacco extract
Fracture site may be opened, treated
Intramuscular pocket created in mid-substance of each side of musculus
longissimus thoracis
Bone morphogenetic protein, w/wo bacteria and metal implant, may be
placed in collagen carrier in each pocket
Collagen carrier with BMP,
bacteria and metal implant
packed into intramuscular pocket
Midline incision
Longissimus thoracis muscle
(Wang et al. Proc Natl Acad Sci USA. 1990;87:2220–4)
(Bonnarens and Einhorn. J Orthop Res. 1984;2:97-101)
ASSESSMENTS
High Resolution Radiography
Micro-Computed Tomography
Volume, surface area and average grayscale of newly mineralized
callus within a segmental defect and bridging the outside of the defect
Radiographic Bony Lysis
Torsional Failure Testing
The number of sites of osteolysis where K-wires cross cortical bone in
infected segmental defects correlates with torsional stiffness of defect
fixation
Histology
Undecalcified histology
Treated and intact femurs loaded to failure in torsion in materials test
machine. Torque versus angular displacement used to compute torque
and energy absorbed to failure, and torsional stiffness of callus.
Bacteriology
Decalcified histology
Osmotic Infusion Mini-Pumps for Drug Delivery
Nicotine and other agents delivered via
continuous infusion
Faxitron radiographs imported into image analysis system; templates
used to measure area of newly formed callus
Qualitative cultures from bacterial
swabs
Quantitative bacteriology - number
of recovered colony-forming units
(CFUs) per gram of bone
Other Assessment Tools
Real-time PCR for measurement of mRNA gene expression
Atomic absorption spectrophotometry for measurement of calcium
content of the decalcifying solution from newly formed callus
INFECTION
Clinical Problem
Infection directly hinders fracture healing
Bacteria colonize surface of fracture fixation device and form impenetrable biofilm
Effective treatment of infection usually requires removal of fixation device
This is counterproductive until fracture has attained stability
An intervention is needed that would stimulate fracture healing in the presence of
infection and colonized hardware, allowing earlier removal of implant and more
effective treatment of infection
Specific Aim of Study
Determine whether bone morphogenetic proteins (BMPs) are capable of stimulating
bone formation in the presence of acute or chronic infection with colonized hardware
Hypotheses
BMP is able to induce new bone to form in an internally-stabilized segmental defect in
the rat femur with acute contamination or chronic infection, and administration of systemic and/or local antibiotic will enhance this effect
Animal Model
Internally stabilized segmental defect in rat femur with acute contamination or chronic infection
METHODS
Experimental Design
400 g Sprague-Dawley rats
4
Defect created, collagen carrier wetted with 10 CFUs Staphylococcus aureus, placed
in defect
After 2 weeks, defect debrided, treated with 0, 20 or 200 µg BMP/carrier ± local
antibiotic ± systemic antibiotic (design table right)
Recombinant human BMPs
- Bone Morphogenetic Protein-2 (BMP-2) in type 1 bovine collagen sponge
- Osteogenic Protein-1 (OP-1) in lyophilized type I bovine collagen
Systemic antibiotic: intramuscular injection of ceftriaxone (50 mg/kg once per day for
28 days)
Local antibiotic: ceramic-collagen matrix carrier wetted with 100 mg cefazolin in
sterile water
When rhBMP-2 + local antibiotic, collagen sponge with rhBMP-2 wrapped around
collagen matrix with antibiotic, placed in defect
Create defect,
Contaminate with S. aureus
Time 0
Debride defect, treat
with BMP, antibiotic
2 weeks
BMP
(Dose)
Treatment
Local antibiotic
(Cefazolin)
200 mg
yes
20 mg
yes
0 mg
yes
200 mg
no
20 mg
no
0 mg
no
Systemic antibiotic
(Ceftriaxone)
yes
no
yes
no
no
yes
no
yes
no
yes
no
Animals euthanized
+ 2, 4, 8 or 12 weeks
after debridement
Assessments
Micro-computed tomography
Undecalcified histology
High resolution radiographs (bony lysis and area of new callus)
Qualitative and quantitative bacteriology
Torsional failure testing
RESULTS
4 weeks
No
Systemic
antibiotic
antibiotic
BMP maintained its osteoinductive capability despite presence
of chronic infection and colonized hardware
Effect of BMP on newly mineralized callus …
- was dose dependent
- was enhanced with systemic antibiotic therapy
- increased with time from debridement
20 µg
rhBMP-2
200 µg
rhBMP-2
12 weeks
No
Systemic
antibiotic
antibiotic
INFECTION
RESULTS - CONTINUED
No substantial callus formed in chronically infected defects without sufficiently high dose of BMP; this was enhanced with systemic antibiotic
4 weeks
No antibiotic
12 weeks
Antibiotic
No antibiotic
Antibiotic
0 µg
rhBMP-2
20 µg
rhBMP-2
200 µg
rhBMP-2
Combination of high dose of BMP with local/systemic antibiotic led
to more new bone than BMP with systemic antibiotic alone
Defect strengths with high dose of BMP with local/systemic antibiotic
were not significantly different than intact contralateral femurs
†
20 or 200 µg rhBMP-2 in
absorbable collagen sponge
L = Local antibiotic
S = Systemic antibiotic
*
*
* Significantly less than 200+L+S (p<0.045)
*
*
*
*
† 12 weeks significantly greater than 2 weeks (p=0.003)
Bony lysis due to chronic infection was less with antibiotic than without,
but was still present over the study period regardless of treatment
* Significantly less than intact contralateral femur (p<0.005)
More new bone bridged the defect with rhOP-1 (below); more bone
formed within defect with rhBMP-2
Regulatory Status of BMPs
REFERENCES
Recombinant human bone morphogenetic protein-2/Absorbable Collagen Sponge has
been approved by FDA for treatment of acute, open tibial fractures stabilized by
intramedullary nail fixation in skeletally mature patients within 14 days of initial
fracture.
OP-1 implant has been approved under a humanitarian device exemption (HDE) for
use in recalcitrant long bone non-unions where autograft is unfeasible and alternative
treatments have failed.
- Chen X, Kidder LS, Lew WD: Osteogenic Protein-1 Induced Bone Formation in an Infected Segmental
Defect in the Rat Femur. Journal of Orthopaedic Research, Vol. 20, No. 1, pp. 142-150, 2002.
- Chen X, Schmidt AH, Tsukayama DT, Bourgeault CA, Lew WD: Recombinant Human Osteogenic
Protein-1 Induces Bone Formation in a Chronically Infected, Internally Stabilized Segmental Defect in the
Rat Femur. Journal of Bone and Joint Surgery [Am], Vol. 88, No. 7, pp. 1510-1523, 2006.
- Chen X, Schmidt AH, Mahjouri S, Polly Jr DW, Lew MD: Union of a Chronically Infected Internally
Stabilized Segmental Defect in the Rat Femur after Debridement and Application of rhBMP-2 and
Systemic Antibiotic. Journal of Orthopaedic Trauma, Vol. 21, No. 10, pp. 693-700, 2007.
TAKE-HOME POINTS
Animal modeling suggests that BMP-2 and OP-1 maintain their osteoinductive capability in the presence of chronic infection and colonized
hardware, and that this property is enhanced by systemic and local antibiotic
This intervention potentially accelerates fracture healing in the presence of infection and colonized fixation device, permitting earlier removal of the
device and more effective treatment of infection
Further animal modeling and clinical studies are warranted to determine BMP dosing and timing, and effect of other pathogens and antibiotics
DIABETES MELLITUS
Clinical Problem
2 weeks
Type 1 diabetes mellitus.....
- Alters properties of bone
- Inhibits fracture healing
- Is associated with increased risk of
nonunion, delayed union, wound necrosis,
and increased incidence of infection
Specific Aim of Study
Determine whether BMP can overcome
inhibition of fracture healing in animal model
of streptozotocin-induced diabetes
4 weeks
Non-diabetic: Sham surgery
Non-diabetic: Sham surgery
Diabetic: Sham surgery
Diabetic: Sham surgery
Diabetic: 0mg BMP + Carrier
Diabetic: 0mg BMP + Carrier
Diabetic: 25mg BMP + Carrier
Diabetic: 25mg BMP + Carrier
Hypothesis
BMP will induce more and stronger callus in diabetic animals with a
fracture, compared to a fracture without BMP
Animal model
Closed fracture in rat femur with streptozotocin-induced diabetes
Experimental design
METHODS
63 Sprague Dawley rats
Subcutaneous injection of 50 mg/kg of streptozotocin in buffer
(diabetic) or buffer only (non-diabetic controls)
After 2 weeks …
- Closed fracture created
- Fracture site surgically opened, treated with 0 or 25 µg
recombinant human BMP (Osteogenic Protein-1) in 50 mg of
collagen carrier
Fracture site opened and immediately closed without treatment in
sham surgery animals
Femurs harvested 2 or 4 weeks post-fracture/treatment
Failure strengths of fractures in diabetic animals with BMP at 4
weeks were not significantly different than intact contralateral
femurs; failure strengths without BMP were significantly less than
intact femurs
*
*†
†
*†
Assessments
Weight
Serum glucose levels (considered diabetic if greater than 300 mg/dl)
High resolution radiographs to measure callus area
Torsional failure testing
* Intact femur significantly greater than corresponding fractured femur (p<0.012)
† Significantly less than intact non-diabetic sham surgery controls (p<0.009)
From 3 to 30 times more radiographic area of new callus with
BMP/carrier than with carrier-only and sham surgery groups
RESULTS
BMP overcame diabetes-induced inhibition of fracture healing by
forming more and stronger new callus in the rat, compared to
fractures with diabetes without BMP
2 weeks
Non-diabetic: Sham surgery
*
*
4 weeks
Non-diabetic: Sham surgery
* Significantly greater than other treatments at the same time point (p<0.001)
Diabetic: Sham surgery
Diabetic: Sham surgery
All animals gained weight over the study; weights with streptozotocin
increased at a lower rate than non-diabetic controls
Diabetic: 0mg BMP + Carrier
Diabetic: 25mg BMP + Carrier
Diabetic: 0mg BMP + Carrier
Diabetic: 25mg BMP + Carrier
REFERENCES
- Kidder LS, Chen X, Lew WD: Bone Morphogenetic Protein Mediated Fracture Healing in the Diabetic Rat.
Transactions of 51st Annual Meeting of the Orthopaedic Research Society, Vol. 30, Poster No. 1098, 2005.
- Kidder LS, Chen X, Schmidt AH, Lew WD. OP-1 Accelerates Fracture Healing in a Rat With StreptozotocinInduced Diabetes. Submitted to Clinical Orthopaedics and Related Research, in review.
TAKE-HOME POINT
Animal modeling suggests that BMP may overcome the osteogenic inhibition of fracture healing by uncontrolled streptozotocin-induced diabetes
SMOKING
Clinical Problem
Smoking
- Inhibits fracture repair and spinal fusion, resulting in delayed union or nonunion
- Increases risk of sustaining a fracture
- Increases risk of osteoporosis
Specific Aim of Study
Determine effects of nicotine and tobacco extract, separately and in combination, on fracture healing
Hypotheses
Tobacco extract, with or without nicotine, reduces fracture healing strength
Nicotine at physiologic doses has no effect on fracture healing strength
Animal Model
Closed femoral fracture model in rat
METHODS
Study 1
Study 2
Effect of nicotine and tobacco extract, separately and together
Effect of increasing dose of nicotine
Nicotine dose
Treatment
No Tobacco Extract
Tobacco Extract
No Nicotine
n=22 (control)
n=23
Treatment
Nicotine: 3 mg/kg/day
n=22
n=22
Number of animals
0
3
mg/kg/day mg/kg/day
n=16
n=19
6
mg/kg/day
12
mg/kg/day
n=19
n=22
Nicotine administration: Osmotic mini-pump implanted subcutaneously (3 mg/kg/day ~ 1-2 packs/day human dose)
Tobacco extract administration: Tobacco-soaked drinking water (15 ng/ml anitabine ~ 1 pack/day human dose)
Control: Saline in mini-pump, regular drinking water
Assessments:
Radiographs
Torsional failure testing
Undecalcified histology
Serum nicotine and cotinine
RESULTS
Study 1
Study 2
Nicotine alone did not impair fracture healing, but tobacco extract with
or without nicotine did
Other tobacco smoke toxins may adversely affect fracture healing
Increased dose of nicotine increased fracture strength
Mean maximum torque (Nm) Mean stiffness (Nm/º)
Mean energy to failure (Nm/º)
*
Nicotine dose: 0 mg/kg
3 mg/kg
6 mg/kg
12 mg/kg
* Significantly lower than both no tobacco extract groups (p<0.023)
Considerations of Model
Continuous rather than bolus application of nicotine and tobacco
Elements in smoke are not included in this model
Rat model representing human fracture healing at specific time points and
dosages
0 mg/kg
0mg/kg
3 mg/kg
3mg/kg
6 mg/kg
6mg/kg
12 mg/kg
12mg/kg
REFERENCES
- Skott M, Andreassen TT, Ulrich-Vinther M, Chen X, Keyler DE, LeSage MG, Pentel PR, Bechtold JE, Soballe K. Tobacco extract but not nicotine impairs the mechanical strength of fracture healing in rats. Journal of Orthopaedic
Research. 2006; 24:1472-1479.
- Friess D, Chen, X, Bourgeault C, Kyle R, Keyler D, Pentel P, LeSage M, Søballe K, Bechtold JE Nicotine does not inhibit fracture repair in rats. Transactions Orthopaedic Research Society, Vol.32, p. 223, San Diego, CA, 2007.
TAKE-HOME POINTS
Constituents of tobacco may be more deleterious than nicotine in delaying fracture healing
High doses of nicotine may stimulate fracture healing
Nicotine patches in smokers may satisfy their addiction while not delaying fracture healing
Smokers with fractures may be safely treated with aggressive nicotine replacement therapy to quit smoking
GLUCOCORTICOID THERAPY
Clinical Problem
28 Days Post-Fracture
Therapeutic glucocorticoids at pharmacologic doses may result in...
- Osteopenia
- Pathologic fracture
- Inhibition of fracture healing with risk of delayed union or
nonunion
No Prednisolone (control)
Carrier only (no BMP)
Prednisolone
Carrier only (no BMP)
No Prednisolone (control)
Carrier with BMP
Prednisolone
Carrier with BMP
Specific Aim of Study
Determine whether BMP can overcome inhibition of fracture healing
from pharmacologic doses of prednisolone
Hypothesis
BMP will overcome prednisolone-induced inhibition of fracture
healing by forming more and stronger callus, compared to fractures
with prednisolone without BMP
Animal model
Closed fracture in rat femur
METHODS
Experimental design
108 Sprague Dawley rats
Surgical placement of subcutaneous time-release prednisolone (2.2
mg/kg/d) or placebo pellets
After 2 weeks...
- Closed fracture created
- Fracture site surgically opened, treated with 0 or 25 µg recombinant
human BMP (Osteogenic Protein-1) in 50 mg of collagen carrier
Femurs harvested at 3, 10, 21, 28 and 42 days post-fracture/treatment
Implantation
of time-release
prednisolone or
placebo pellets
Healing fractures 67% stronger at 28 days post-fracture with
BMP/carrier than with carrier only
†
†
Creation of closed fracture;
treatment of fracture site
*
+3 days
+10 days
+21 days
+28 days
Day 0
+42 days
Day 14
Assessments
High resolution radiographs and image analysis system to measure
area of newly mineralized callus
Histomorphometry using decalcified mid-sagittal sections
Torsional failure testing
* Fractured significantly weaker than intact (p<0.007)
† BMP/carrier significantly greater than carrier only (p<0.043)
Nearly 2.5 times more radiographic area of newly mineralized callus
at 28 days post-fracture with BMP/carrier than with carrier only
†
RESULTS
BMP overcame prednisolone-induced inhibition of fracture healing by
forming more and stronger callus in the rat, compared to fractures with
prednisolone without BMP
28 Days Post-Fracture
*
No Prednisolone (control)
Carrier only (no BMP)
Prednisolone
Carrier only (no BMP)
No Prednisolone (control)
Carrier with BMP
Prednisolone
Carrier with BMP
* Significantly greater than carrier only ± prednisolone (p<0.001)
† Significantly greater than all other treatments (p<0.028)
REFERENCES
- Gilley RS, Wallace LJ, Bourgeault CA, Kidder LS, Chen X, Bechtold JE. Influence of bone morphogenetic
protein on glucocorticoid-inhibited fracture healing in a closed femoral fracture rat model. 49th Annual Meeting of
the Orthopaedic Research Society, 2003, Poster #502, p 49, New Orleans, LA.
- Gilley RS, Wallace LJ, Bourgeault CA, Kidder LS, Bechtold JE. Glucocorticoid effects on fracture healing using
a rat closed femoral fracture model. 30th Ann Conf Vet Orthop Soc, p. 86, 2003, Steamboat Springs, CO.
TAKE-HOME POINT
Animal modeling suggests that BMP may overcome prednisolone-induced inhibition of fracture healing