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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