TOWARDS THE CREATION OF POLYMER COMPOSITES WHICH CAN BE REFILLED WITH ANTIBIOTICS AFTER IMPLANTATION FOR INFECTION TREATMENT By ERIKA LEAH CYPHERT Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Horst von Recum, Ph.D. Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY January 2021 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Erika Leah Cyphert Candidate for the Doctor of Philosophy degree*. (signed) Steven Eppell, Ph.D. (chair of committee) Horst von Recum, Ph.D. Eben Alsberg, Ph.D. Agata Exner, Ph.D. Jonathan Pokorski, Ph.D. (date) September 25, 2020 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 To my grandparents with love – Phil and Ann Cyphert Ted and Dorothy Lippold Florence Miller 3 TABLE OF CONTENTS TABLE OF CONTENTS………………………………………………………………..4 LIST OF TABLES…………………………………………………………………..….10 LIST OF FIGURES…………………………………………………………………….13 LIST OF ABBREVIATIONS……………………………………………………….…21 ACKNOWLEDGEMENTS……………………………………………………………24 ABSTRACT…………………………………………………………………………..…27 CHAPTER 1: DIAGNOSIS AND BIOMATERIAL-BASED TREATMENTS FOR PERIPROSTHETIC JOINT INFECTIONS………………………………………….29 1.1. LIMITATIONS OF CLINICAL TREATMENT OF PERIPROSTHETIC INFECTION…………………………………………………………………30 1.1.1. INTRODUCTION…………………………………………….…30 1.1.2. ISOLATION OF MICROBIAL ORGANISMS………………....33 1.1.3. POSSIBLE UNDERLYING PATIENT COMORBIDITIES……33 1.1.4. BIOFILM FORMATION AND BACTERIAL RESISTANCE…34 4 1.1.5. REVISION PROCEDURES/INITIAL TREATMENT FAILURES....................................................................................36 1.1.6. SUMMARY……………………………………………………...37 1.2. NOVEL TREATMENT MODALITIES FOR PJIS………………………...38 1.2.1. LIMITATIONS WITH TRADITIONAL ANTIBIOTIC-LADEN PMMA BONE CEMENT……………………………………..…38 1.2.2. COMMERCIALLY AVAILABLE ALTERNATIVE BIOMATERIALS FOR ANTIBIOTIC-LADEN PMMA BONE CEMENT………………………………………………………...40 1.2.3. TITANIUM NANOTUBE ARRAYS……………………………41 1.2.4. IMPLANT COATINGS WITH ANTIBACTERIAL EFFECT….42 1.2.5. POLYMERS/HYDROGELS…………………………………….43 1.2.6. CYCLODEXTRIN (CD)-BASED DRUG DELIVERY………...44 1.2.7. CONCLUSIONS………………………………………………...48 1.3. ACKNOWLEDGEMENTS…………………………………………....……48 5 CHAPTER 2: ANTIBIOTIC REFILLING OF PMMA BONE CEMENT THROUGH INCORPORATION OF CYCLODEXTRIN FOR TREATMENT OF PERIPROSTHETIC JOINT INFECTION…………………………………………...49 2.1. ABSTRACT…………………………………………………………………50 2.2. INTRODUCTION…………………………………………………………..51 2.3. MATERIALS AND METHODS……………………………………………55 2.4. RESULTS…………………………………………………………………...62 2.5. DISCUSSION……………………………………………………………….75 2.6. CONCLUSIONS……………………………………………………………79 2.7. ACKNOWLEDGEMENTS…………………………………………………80 CHAPTER 3: TREATMENT OF BROAD-SPECTRUM PERIPROSTHETIC JOINT INFECTIONS USING COMBINATORIAL ANTIBIOTIC PMMA COMPOSITE…………………………………………………………………...………81 3.1. ABSTRACT…………………………………………………………………82 3.2. INTRODUCTION…………………………………………………………..83 3.3. MATERIALS AND METHODS……………………………………………87 3.4. RESULTS AND DISCUSSION………………………………………….…91 6 3.5. CONCLUSIONS…………………………………………………………..115 3.6. ACKNOWLEDGEMENTS………………………………………………..116 CHAPTER 4: POLYMERIZED CYCLODEXTRIN CAN BE REFILLED WITH ANTIBIOTICS IN THE PRESENCE OF BACTERIAL BIOFILMS…………….117 4.1. ABSTRACT……………………………………………………………..…118 4.2. INTRODUCTION…………………………………………………………119 4.3. MATERIALS AND METHODS…………………………………………..122 4.4. RESULTS AND DISCUSSION…………………………………………...126 4.5. CONCLUSIONS…………………………………………………………..135 4.6. ACKNOWLEDGEMENTS………………………………………………..136 CHAPTER 5: ANTIBIOTIC REFILLING OF PMMA BONE CEMENT COMPOSITE THROUGH BONE AND MUSCLE TISSUE TO TREAT PERIPROSTHETIC JOINT INFECTIONS………………………………………...137 5.1. ABSTRACT………………………………………………………………..137 5.2. INTRODUCTION…………………………………………………………138 5.3. MATERIALS AND METHODS………………………………………..…141 5.4. RESULTS AND DISCUSSION…………………………………………...148 7 5.5. CONCLUSIONS…………………………………………………………..167 5.6. ACKNOWLEDGEMENTS………………………………………………..168 CHAPTER 6: PROCESSING TECHNIQUE OF PMMA BONE CEMENT COMPOSITE DICTATES REFILLING CAPACITY FOR TREATMENT OF PERIPROSTHETIC JOINT INFECTIONS………………………………………...169 6.1. ABSTRACT………………………………………………………………..170 6.2. INTRODUCTION…………………………………………………………171 6.3. MATERIALS AND METHODS…………………………………………..174 6.4. RESULTS………………………………………………………………….179 6.5. DISCUSSION……………………………………………………………...199 6.6. CONCLUSIONS…………………………………………………………..205 6.7. ACKNOWLEDGEMENTS………………………………………………..206 CHAPTER 7: CONCLUSIONS AND FUTURE DIRECTIONS…………………..207 7.1. CONCLUSIONS…………………………………………………………..207 7.2. FUTURE DIRECTIONS…………………………………………………..219 7.3. ACKNOWLEDGEMENTS………………………………………………..227 APPENDIX…………………………………………………………………………….228 8 PERMISSIONS………………………………………………………………...228 REFERENCES………………………………………………………………………...243 9 LIST OF TABLES TABLE 2-1 Mechanical testing of cylindrical PMMA-CD composite samples ultimate compressive strength, work to peak load, and strain to peak load…....................................................................................69 TABLE 2-2 Quantification of RMP refilling from agarose refilled polymer samples: pure PMMA versus β-CD disks (top) and pure PMMA versus PMMA-CD composites with 5 and 10 wt% CD microparticles (bottom)…………………………………………..72 TABLE 3-1 Quantification of various parameters of cylindrical PMMA-CD composite samples containing either only gentamicin or tobramycin (control), 10 wt% empty (nondrug-filled) CD microparticles with gentamicin or tobramycin, and 10 wt% RMP- filled CD microparticles with gentamicin or tobramycin that were micro-CT scanned……………………………………………....100 TABLE 3-2 Quantification of mechanical properties of PMMA-CD cylindrical composite samples containing either (a) tobramycin or gentamicin alone (controls), (b) 10 wt% empty (nondrug-filled) CD microparticles with either free gentamicin or tobramycin, or (c) 10 wt% RMP-filled CD microparticles with either free gentamicin or tobramycin……………………………………………..…….…105 10 TABLE 3-3 Quantification of amount of RMP initially filled (top) and amount of RMP refilled through agarose phantom model into PMMA-CD composite bead samples containing either 5 or 10 wt% empty (nondrug-filled) CD microparticles with either free gentamicin or tobramycin (bottom)……………………………………………109 TABLE 4-1 Mass of antibiotics filled into CD and dextran polymer disks in agarose phantom model………………………………………...128 TABLE 4-2 Bacterial quantification of CD and dextran polymer disks with mature biofilms…………………………………………………131 TABLE 6-1 Micro-CT quantification of porosity for hand- and vacuum-mixed PMMA-CD composite cylinders containing either no microparticles or 10 wt% or 15 wt% empty (nondrug-filled) CD microparticles…………………………………………………...186 TABLE 6-2 Quantification of mechanical properties for hand- and vacuum- mixed PMMA-CD composites containing either no CD microparticles or 10 or 15 wt% empty (nondrug-filled) or RMP- filled CD microparticles………………………………………...190 11 TABLE 7-1 Summary of relationship of amount of drug injected into refilling model and amount filled into PMMA-CD composite to the duration of antimicrobial activity possible…………………..…210 TABLE 7-2 Analysis of impact of parameters controlling mechanical strength and refilling capacity of PMMA-CD composite……………..…214 12 LIST OF FIGURES FIGURE 1-1 Schematic depicting challenges and downstream effects of inappropriate periprosthetic joint infection diagnosis……………31 FIGURE 1-2 Proliferation of a single resistant bacterium when repeatedly treated with antibiotics……………………………………..…….36 FIGURE 1-3 Free-radical polymerization of PMMA bone cement upon combination of liquid (methyl methacrylate monomer) and powdered component (benzoyl peroxide initiator and pre- polymerized PMMA beads)…………………………………..….38 FIGURE 1-4 Interior of antibiotic-laden PMMA before and after releasing some antibiotic from the sample……………………………………….39 FIGURE 1-5 Structure of soluble CD monomer, soluble pre-polymerized CD, and cross-linked insoluble CD polymer microparticles….………44 FIGURE 1-6 Antibiotic refilling of PMMA versus cross-linked CD polymer when implanted in soft tissue…………………………………….45 FIGURE 1-7 Daily release profile from cyclodextrin (CD) affinity-based polymers as compared to drug release through diffusion (i.e. PMMA). Polymers that use affinity-based release may be refilled after implantation (b)…………………………………………….46 13 FIGURE 2-1 Schematic outlining synthesis of PMMA-CD composite and subsequent evaluations…………………………………………...55 FIGURE 2-2 Zone of inhibition study of antimicrobial activity of three antibiotics (RMP, tobramycin, and gentamicin) freely added into PMMA (plain) beads against S. aureus………………………….63 FIGURE 2-3 Zone of inhibition study of antimicrobial activity of antibiotic- filled (RMP, tobramycin, and gentamicin) CD microparticles as PMMA-CD composite beads against S. aureus………………….64 FIGURE 2-4 Drug release profiles and antimicrobial susceptibility zone of inhibition testing with a) tobramycin and b) gentamicin freely added into PMMA beads and from c-g) antibiotic-CD microparticles in PMMA-CD composite beads………………….66 FIGURE 2-5 Representative compressive load-displacement curves of PMMA- CD composite cylinders. Load-displacement curves of all groups superimposed (left) and zoomed in curve of free RMP cement (right)………………………………………………………….…68 FIGURE 2-6 Image of PMMA beads (no CD microparticles) and CD disks prior to RMP refilling in agarose model (top) and image of polymer 14 samples 48 hours after being refilled with RMP in model (bottom)…………………………………………………………..71 FIGURE 2-7 Image of RMP refilling