- Email: [email protected]
Locally delivered minocycline microspheres do not impair osseointegration of titanium implants in a rat femur model
Journal of Orthopaedics 20 (2020) 213–216
Contents lists available at ScienceDirect
Journal of Orthopaedics journal homepage: www.elsevier.com/locate/jor
Original Article
Locally delivered minocycline microspheres do not impair osseointegration of titanium implants in a rat femur model
T
Joshua A. Shapiro (MD)a,∗, Samuel AbuMoussa (MD)a, Christopher P. Lindsay (MD)a, Gabriel B. Mason (BA)a, Laurence E. Dahners (MD)a, Paul S. Weinhold (PhD)a,b,c a
Department of Orthopaedics, University of North Carolina, Chapel Hill, NC, USA Joint Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC, USA c North Carolina State University, Raleigh, NC, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Minocycline Osseointegration Pushout testing Bone volume fraction Sprague-Dawley
Background: The purpose of this study was to determine whether intramedullary administration of extendedrelease minocycline microspheres would affect osseointegration. Methods: Twenty-two rats were randomized to minocycline or saline femoral intramedullary injection followed by implantation of titanium alloy rods. Following euthanasia at four-weeks, pushout testing was performed and bone-volume-fraction assessed. Results: Pushout strength was marginally greater in minocycline-treated implants (122.5 ± 39.1 N) compared to saline (96.9 ± 26.1 N) (P = 0.098). No difference was observed in energy to maximum load, mean stiffness, or peri-implant bone-volume-fraction (P > 0.05). Conclusions: Peri-implant minocycline administration did not impair implant fixation strength or peri-implant bone-volume, supporting its potential utility as an adjunct to intramedullary implants.
1. Introduction Septic and aseptic loosening remains a challenge in cementless fixation of orthopaedic implants. Methods focusing on mechanical properties of implants have given way to altering biology for improved osseointegration. Ideally, implant coating would both enhance osseointegration and reduce periprosthetic infection. The pathway of osteolysis begins with inflammation. Osteoblasts are stimulated by wear particles to produce pro-inflammatory interleukins, cytokines, and ligands to promote osteoclastic maturation. TNF-α, RANK-L, IFN-y, matrix metalloproteinases (MMP) and metabolites from the 5-lipoxegenase (5-LO) pathway have been found to be involved in osteolysis.1,2 Inhibition of the 5-LO pathway may cause a shunt increase in COX-2 and therefore prostaglandins which have been shown to increase bone formation.2 This effect may also lessen osteolysis as 5-LO metabolites have been shown to inhibit bone formation in vitro. Specifically, the peptide-leukotrienes LTC, LTD, and LTE, 5-hydroperoxyeicosatetraenoic acid (5-HETE), and leukotriene B (LTB) have been found to directly and indirectly induce bone resorption.2 Furthermore, inhibition of MMP activity has decreased resorption in an oophorectomized rat femur model.3 Thus, targeting of pro-inflammatory peptides and cytokines may reduce osteolysis, driving an ∗
increased yield of osseointegration and decreased aseptic loosening. Minocycline, in addition to having potent antimicrobial effects through inhibition of bacterial protein synthesis, demonstrates antiinflammatory properties via inhibition of 5-LO and MMPs4 and may be beneficial for augmenting osseointegration, although this has not yet been studied. Minocycline is presently available for periodontal use in a microsphere formulation made of resorbable glycolide-lactide slow-release polymer that has been shown to be effective in the management of chronic periodontitis at concentrations of 2%.5 The polymer is approved by the US Food and Drug Administration, is considered nontoxic, and displays constant and controlled release kinetics over 12–26 days.6 It has not yet been determined if this minocycline microsphere encapsulation could be used as a metal coating or injected in the intramedullary space for slow release during the pivotal early phases of osseointegration and infection susceptibility; however, its potent antiinflammatory properties suggest potential. The purpose of this study was to determine if local intramedullary delivery of minocycline microspheres could enhance osseointegration in a distal femoral rat model. With the theoretical osteoinductive properties of minocycline, we hypothesized that local administration of minocycline microspheres would enhance osseointegration in the form
Corresponding author. University of North Carolina Department of Orthopaedics, 130 Mason Farm Rd #3155, Chapel Hill, NC, 27599, USA. E-mail address: [email protected] (J.A. Shapiro).
https://doi.org/10.1016/j.jor.2019.12.007 Received 26 November 2019; Accepted 10 December 2019 Available online 12 December 2019 0972-978X/ © 2019 Professor P K Surendran Memorial Education Foundation. Published by Elsevier B.V. All rights reserved.
Journal of Orthopaedics 20 (2020) 213–216
J.A. Shapiro, et al.
implant stiffness (N/mm), were calculated from the load-displacement curves.
of mechanical stability of the implant and peri-implant bone volume. 2. Materials and methods
2.4. μCT analysis Approval was obtained from the local institutional animal care and use committee prior to implementation of study procedures. Twentytwo female Sprague-Dawley rats at age 24-weeks (Envigo Inc., Dublin, VA) were selected and randomized to match weight and assigned to either the minocycline or saline placebo group. Rats were housed in pairs in an officially registered and accredited USDA Animal Research facility and allowed ad libitum access to food and water with a 12-hr light/dark cycle (7am-7pm) throughout the duration of the study.
Right femurs were scanned using a specimen μCT system (Model μCT 40, Scanco Medical, Brüttisellen, Switzerland) with a 10 mm field of view on medium resolution with a voxel size of 16 μm along a 6 mm length 1.5 mm from the distal end of the implant.8 The X-ray power setting was 70 kVp, 114 μA, and 8 W. The scans had an integration time of 300 m s and were averaged once. The resulting μCT scans were analyzed using software developed for processing medical images (Image J, National Institutes of Health, Bethesda, Maryland). The implant was dilated by five pixels (80 μm) to exclude the metal-induced artifact.9 The bone was segmented out using a low and high threshold of 529 and 1615 mgHA/cm3 respectively. The implants were segmented using a threshold ≥2249 mgHA/cm3. The BV/TV within 500 μm of the implant was calculated. A cylinder was constructed in the femur that had a diameter 1000 μm larger than the mean diameter of the implant. The volume of bone within this region was divided by the volume of the cylinder minus the dilated implant volume within the cylinder. The resulting percentage represented the medullary BV/TV.
2.1. Titanium implant preparation Implants were fabricated using Grade 5 Titanium (Ti–6Al–4V) and produced using direct metal laser sintering on an EOS M280 15° (EOS GmbH. Krailling, Germany) from vertical with beam offset of 0.09 mm using a powder size ranging from 25 to 45 μm. The titanium rods were produced to 20 mm long and 1.2 mm in diameter with a dimple in each end to facilitate surgical implantation and mechanical pushout. The “as built” surface was textured with surface roughness of Ra = 7.7 ± 1.8 μm. All implants were textured by acid etching in a 48% sulfuric acid bath at 60 °C while agitating with a stir bar for 30 min. The implants were rinsed in deionized water, cleaned in deionized water for 10 min under ultrasonic agitation, dehydrated in a 70% ethanol solution, and allowed to air dry before packaging for sterilization by autoclave. This procedure is similar to what has been done previously in this model.7
2.5. Statistical analysis Treatment differences in the outcome measure were evaluated by unpaired Student's t-test using a statistical analysis program (SigmaPlot v11.0, Systat Software, San Jose, CA). A significance level of 0.05 was used for detecting treatment effects. A power analysis prior to the study had determined that a sample size of 11 animals per group would be required to detect a 42% change in the pushout fixation strength for a power of 0.80 and significance level of 0.05 assuming a standard deviation of 30% of the control mean.
2.2. Surgical implantation and treatment delivery Under isoflurane anesthesia, a 2 cm longitudinal incision was made on the anteromedial aspect of the rat's knee and the patella was reflected laterally to expose the intercondylar notch of the femur. An 18gauge needle was inserted in retrograde fashion into the medullary canal of the femur, which was then reamed with a 1 mm hand-drill bit to the level of the lesser trochanter. 0.1 mL of normal saline or 0.1 ml of minocycline microspheres (1 mg/ml; OraPharma) was injected directly into the medullary cavity under direct visualization followed by immediate implantation of the titanium alloy implant. The implant was seated flush with the articular surface of the distal femur. The joint capsule was closed with absorbable suture and skin incisions closed with wound clips. The procedure was repeated for the contralateral femur. Seven days post-operatively the wound clips were removed and radiographs of both femurs were taken to ensure proper placement of the implants. The animals were allowed to heal for four-weeks, at which time they were euthanized and femurs were isolated for biomechanical testing and μCT. Following euthanasia, femurs were carefully removed and denuded to bone. The left femurs were wrapped in 0.9% salinesolution-soaked gauze and frozen at −20 °C until biomechanical testing. The right femurs were fixed in neutral buffered formalin for 48 h and transferred to 70% ethanol in preparation for μCT analysis.
3. Results Rats were 24-weeks old at the onset of the study. Mean body weights of the groups did not differ and were the following: control (392.3 ± 9.5 g) and minocycline (393.3 ± 7.9 g). 3.1. Biomechanical testing Pushout testing to failure revealed that the mean force required to pushout minocycline (122.5 ± 39.1 N) treated implants was 26% greater than the control (96.9 ± 26.1 N). However, this difference did not reach statistical significance (p = 0.098). Similarly, the energy to maximum load was slightly greater for minocycline treated implants. However, there was no significant difference in this measure between the groups. The mean stiffness was similar in both groups (p = 0.923) (Fig. 1). 3.2. μCT analysis μCT analysis determined the mean peri-implant bone volume fraction (BV/TV) of the minocycline (36.7 ± 10.8) trended to higher than the control group (30.6 ± 7.5); however, the difference was not significantly different (p = 0.182) (Fig. 2).
2.3. Biomechanical testing Prior to testing, each femur was thawed to room temperature. Once thawed, femurs were prepared by exposing the proximal end of the implant via careful sharp excision of the proximal femur until the implant was identified. The distal end of the femur was potted in a polymethylmethacrylate dental cement (Ortho Jet BCA, Lang Dental, Wheeling, IL) and the implant was subjected to a pushout test using a material testing system (8500 Plus, Instron Corp., Norwood, MA). The long axis of the implant was aligned with the axis of push-out loading and loaded at 2 mm/min until failure of the bone-implant interface was reached. Maximum push-out load (N), energy to failure (mJ), and bone-
4. Discussion The results of the present study suggest that local administration of slow release minocycline did not adversely affect osseointegration, and instead resulted in a trend toward enhanced osseointegration, suggesting that local delivery of minocycline can be used safely as a periimplant adjuvant for infection prophylaxis or the treatment of prosthetic joint infection (PJI) without compromising osseointegration. This 214
Journal of Orthopaedics 20 (2020) 213–216
J.A. Shapiro, et al.
Fig. 1. Box plots of results from push-out testing. Dashed lines represent the mean, solid lines represent the median, and tick marks represent the 5%/95% confidence intervals.
osteolysis and infection are chronic issues in arthroplasty that cannot be addressed fully during the 4-week immediate postoperative period; however, this study does address acute osseointegration failure. Furthermore, the routine use of local antibiotics for osseointegration enhancement or infection prophylaxis is controversial and may increase the prevalence of resistant organisms. Analysis in an early infection model is now warranted to investigate the combined antimicrobial and osseointegration utility of locally delivered minocycline microspheres to orthopaedic implants, as this study supports that its use would not be harmful. 5. Conclusion Peri-implant application of minocycline did not have a deleterious effect on implant fixation strength or peri-implant bone volume in our murine model, supporting that minocycline microspheres could be applied to an intramedullary implant without compromising osseointegration.
Fig. 2. Box plot of peri-implant bone volume fraction of the metaphyseal cancellous bone of the proximal femur.
is not the first time that an antibiotic has been studied for osseointegration; however, it is the first study of its kind to locally administer minocycline with a slow release delivery system that is already available for commercial use in periodontal practice. The initial dosing was designed to produce an intramedullary concentration of minocycline of 10 μg/ml – a level previously shown to not adversely influence osteoblast cell number or alkaline phosphatase activity in vitro10 – with a subsequent controlled release over the duration of the study period. While the study cannot suggest enhancement in osseointegration, it is possible that increasing or decreasing the dose may have enhanced or impeded osseointegration. Applying agents locally to enhance osseointegration is not new. Incorporating porosity in metallic implants has been shown to enhance osseointegration via bony ingrowth and can create a modulus similar to cancellous bone resulting in excellent biocompatibility.11 Coating of implants with calcium-phosphate analogues and bisphosphonates may reduce aseptic loosening.12 Furthermore, implant coating with antimicrobials covalently tethered to implants may reduce septic loosening. Erythromycin has been shown to reduce osteolysis via inhibition of inflammatory pathways and is routinely a major constituent of successful antibiotic impregnated cements.13 Prior studies have shown that local delivery of erythromycin via coating to Peri-Apatite-coated titanium implants may reduce osteolysis.14 Although controversial, local delivery of antibiotics, has been shown to reduce the rate of PJI.13 The addition of minocycline to the implant or intramedullary space would expand antimicrobial coverage to include methicillin-resistant Staphylococcus aureus which is a major pathogen in acute arthroplasty infections,15 while producing an anti-inflammatory state that at least in this study trended toward statistically significant enhanced osseointegration. There are limitations to this study. Significance was not met despite a trend in a positive difference. However, an a priori power analysis suggested adequate sample size to see a difference. Additionally,
Author contributions JAS: data analysis/interpretation, drafting the manuscript, and editing the manuscript. SA: experimental design, data collection, and editing the manuscript. CPL: experimental design, data collection, and editing the manuscript. GBM: implementation of study methods, data collection, drafting the manuscript, and editing the manuscript. LED: experimental design, senior advisor, and editing the manuscript. PSW: experimental design, implementation of study methods, data analysis/interpretation, and editing the manuscript. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Funding for this study was provided by the Aileen Stock Research Fund, and μCT imaging service was provided by the Small Animal Imaging Facility at the UNC Biomedical Imaging Research Center, which is supported in part by an NCI cancer core grant, P30-CA01608640. References 1. Drees P, Eckardt A, Gay RE, Gay S, Huber LC. Mechanisms of disease: molecular insights into aseptic loosening of orthopedic implants. Nat Clin Pract Rheumatol.
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9. Liu S, Broucek J, Virdi AS, Sumner DR. Limitations of using micro-computed tomography to predict bone-implant contact and mechanical fixation. J Microsc. 2012;245(1):34–42. https://doi.org/10.1111/j.1365-2818.2011.03541.x. 10. Rathbone CR, Cross JD, Brown KV, Murray CK, Wenke JC. Effect of various concentrations of antibiotics on osteogenic cell viability and activity. J Orthop Res. 2011;29(7):1070–1074. https://doi.org/10.1002/jor.21343. 11. Unger AS, Lewis RJ, Gruen T. Evaluation of a porous tantalum uncemented acetabular cup in revision total hip arthroplasty: clinical and radiological results of 60 hips. J Arthroplast. 2005;20(8):1002–1009. https://doi.org/10.1016/j.arth.2005.01. 023. 12. Goodman SB, Yao Z, Keeney M, Yang F. The future of biologic coatings for orthopaedic implants. Biomaterials. 2013;34(13):3174–3183. https://doi.org/10.1016/j. biomaterials.2013.01.074. 13. Springer BD, Lee G-C, Osmon D, Haidukewych GJ, Hanssen AD, Jacofsky DJ. Systemic safety of high-dose antibiotic-loaded cement spacers after resection of an infected total knee arthroplasty. Clin Orthop Relat Res. 2004;427:47–51. 14. Ren W, Wu B, Peng X, et al. Erythromycin inhibits wear debris-induced inflammatory osteolysis in a murine model. J Orthop Res. 2006;24(2):280–290. https://doi.org/10. 1002/jor.20004. 15. Ruhe JJ, Menon A. Tetracyclines as an oral treatment option for patients with community onset skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2007;51(9):3298–3303. https://doi.org/10.1128/AAC.00262-07.
2007;3(3):165–171. https://doi.org/10.1038/ncprheum0428. 2. Cottrell JA, Keshav V, Mitchell A, O'Connor JP. Local inhibition of 5-lipoxygenase enhances bone formation in a rat model. Bone Joint Res. 2013;2(2):41–50. https:// doi.org/10.1302/2046-3758.22.2000066. 3. Williams S, Barnes J, Wakisaka A, Ogasa H, Liang CT. Treatment of osteoporosis with MMP inhibitors. Ann N Y Acad Sci. 1999;878:191–200. 4. Leite LM, Carvalho AGG, Ferreira PLFT, et al. Anti-inflammatory properties of doxycycline and minocycline in experimental models: an in vivo and in vitro comparative study. Inflammopharmacology. 2011;19(2):99–110. https://doi.org/10.1007/ s10787-011-0077-5. 5. Vandekerckhove BN, Quirynen M, van Steenberghe D. The use of locally delivered minocycline in the treatment of chronic periodontitis. A review of the literature. J Clin Periodontol. 1998;25(11 Pt 2):964–968 discussion 978-979. 6. Srirangarajan S, Mundargi RC, Ravindra S, Setty SB, Aminabhavi TM, Thakur S. Randomized, controlled, single-masked, clinical study to compare and evaluate the efficacy of microspheres and gel in periodontal pocket therapy. J Periodontol. 2011;82(1):114–121. https://doi.org/10.1902/jop.2010.100324. 7. Kuroda S, Virdi AS, Li P, Healy KE, Sumner DR. A low-temperature biomimetic calcium phosphate surface enhances early implant fixation in a rat model. J Biomed Mater Res A. 2004;70(1):66–73. https://doi.org/10.1002/jbm.a.30062. 8. Ruppert DS, Harrysson OLA, Marcellin-Little DJ, Abumoussa S, Dahners LE, Weinhold PS. Osseointegration of coarse and fine textured implants manufactured by electron beam melting and direct metal laser sintering. 3D Print Addit Manuf. 2017;4(2):91–97. https://doi.org/10.1089/3dp.2017.0008.
216
Contents lists available at ScienceDirect
Journal of Orthopaedics journal homepage: www.elsevier.com/locate/jor
Original Article
Locally delivered minocycline microspheres do not impair osseointegration of titanium implants in a rat femur model
T
Joshua A. Shapiro (MD)a,∗, Samuel AbuMoussa (MD)a, Christopher P. Lindsay (MD)a, Gabriel B. Mason (BA)a, Laurence E. Dahners (MD)a, Paul S. Weinhold (PhD)a,b,c a
Department of Orthopaedics, University of North Carolina, Chapel Hill, NC, USA Joint Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC, USA c North Carolina State University, Raleigh, NC, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Minocycline Osseointegration Pushout testing Bone volume fraction Sprague-Dawley
Background: The purpose of this study was to determine whether intramedullary administration of extendedrelease minocycline microspheres would affect osseointegration. Methods: Twenty-two rats were randomized to minocycline or saline femoral intramedullary injection followed by implantation of titanium alloy rods. Following euthanasia at four-weeks, pushout testing was performed and bone-volume-fraction assessed. Results: Pushout strength was marginally greater in minocycline-treated implants (122.5 ± 39.1 N) compared to saline (96.9 ± 26.1 N) (P = 0.098). No difference was observed in energy to maximum load, mean stiffness, or peri-implant bone-volume-fraction (P > 0.05). Conclusions: Peri-implant minocycline administration did not impair implant fixation strength or peri-implant bone-volume, supporting its potential utility as an adjunct to intramedullary implants.
1. Introduction Septic and aseptic loosening remains a challenge in cementless fixation of orthopaedic implants. Methods focusing on mechanical properties of implants have given way to altering biology for improved osseointegration. Ideally, implant coating would both enhance osseointegration and reduce periprosthetic infection. The pathway of osteolysis begins with inflammation. Osteoblasts are stimulated by wear particles to produce pro-inflammatory interleukins, cytokines, and ligands to promote osteoclastic maturation. TNF-α, RANK-L, IFN-y, matrix metalloproteinases (MMP) and metabolites from the 5-lipoxegenase (5-LO) pathway have been found to be involved in osteolysis.1,2 Inhibition of the 5-LO pathway may cause a shunt increase in COX-2 and therefore prostaglandins which have been shown to increase bone formation.2 This effect may also lessen osteolysis as 5-LO metabolites have been shown to inhibit bone formation in vitro. Specifically, the peptide-leukotrienes LTC, LTD, and LTE, 5-hydroperoxyeicosatetraenoic acid (5-HETE), and leukotriene B (LTB) have been found to directly and indirectly induce bone resorption.2 Furthermore, inhibition of MMP activity has decreased resorption in an oophorectomized rat femur model.3 Thus, targeting of pro-inflammatory peptides and cytokines may reduce osteolysis, driving an ∗
increased yield of osseointegration and decreased aseptic loosening. Minocycline, in addition to having potent antimicrobial effects through inhibition of bacterial protein synthesis, demonstrates antiinflammatory properties via inhibition of 5-LO and MMPs4 and may be beneficial for augmenting osseointegration, although this has not yet been studied. Minocycline is presently available for periodontal use in a microsphere formulation made of resorbable glycolide-lactide slow-release polymer that has been shown to be effective in the management of chronic periodontitis at concentrations of 2%.5 The polymer is approved by the US Food and Drug Administration, is considered nontoxic, and displays constant and controlled release kinetics over 12–26 days.6 It has not yet been determined if this minocycline microsphere encapsulation could be used as a metal coating or injected in the intramedullary space for slow release during the pivotal early phases of osseointegration and infection susceptibility; however, its potent antiinflammatory properties suggest potential. The purpose of this study was to determine if local intramedullary delivery of minocycline microspheres could enhance osseointegration in a distal femoral rat model. With the theoretical osteoinductive properties of minocycline, we hypothesized that local administration of minocycline microspheres would enhance osseointegration in the form
Corresponding author. University of North Carolina Department of Orthopaedics, 130 Mason Farm Rd #3155, Chapel Hill, NC, 27599, USA. E-mail address: [email protected] (J.A. Shapiro).
https://doi.org/10.1016/j.jor.2019.12.007 Received 26 November 2019; Accepted 10 December 2019 Available online 12 December 2019 0972-978X/ © 2019 Professor P K Surendran Memorial Education Foundation. Published by Elsevier B.V. All rights reserved.
Journal of Orthopaedics 20 (2020) 213–216
J.A. Shapiro, et al.
implant stiffness (N/mm), were calculated from the load-displacement curves.
of mechanical stability of the implant and peri-implant bone volume. 2. Materials and methods
2.4. μCT analysis Approval was obtained from the local institutional animal care and use committee prior to implementation of study procedures. Twentytwo female Sprague-Dawley rats at age 24-weeks (Envigo Inc., Dublin, VA) were selected and randomized to match weight and assigned to either the minocycline or saline placebo group. Rats were housed in pairs in an officially registered and accredited USDA Animal Research facility and allowed ad libitum access to food and water with a 12-hr light/dark cycle (7am-7pm) throughout the duration of the study.
Right femurs were scanned using a specimen μCT system (Model μCT 40, Scanco Medical, Brüttisellen, Switzerland) with a 10 mm field of view on medium resolution with a voxel size of 16 μm along a 6 mm length 1.5 mm from the distal end of the implant.8 The X-ray power setting was 70 kVp, 114 μA, and 8 W. The scans had an integration time of 300 m s and were averaged once. The resulting μCT scans were analyzed using software developed for processing medical images (Image J, National Institutes of Health, Bethesda, Maryland). The implant was dilated by five pixels (80 μm) to exclude the metal-induced artifact.9 The bone was segmented out using a low and high threshold of 529 and 1615 mgHA/cm3 respectively. The implants were segmented using a threshold ≥2249 mgHA/cm3. The BV/TV within 500 μm of the implant was calculated. A cylinder was constructed in the femur that had a diameter 1000 μm larger than the mean diameter of the implant. The volume of bone within this region was divided by the volume of the cylinder minus the dilated implant volume within the cylinder. The resulting percentage represented the medullary BV/TV.
2.1. Titanium implant preparation Implants were fabricated using Grade 5 Titanium (Ti–6Al–4V) and produced using direct metal laser sintering on an EOS M280 15° (EOS GmbH. Krailling, Germany) from vertical with beam offset of 0.09 mm using a powder size ranging from 25 to 45 μm. The titanium rods were produced to 20 mm long and 1.2 mm in diameter with a dimple in each end to facilitate surgical implantation and mechanical pushout. The “as built” surface was textured with surface roughness of Ra = 7.7 ± 1.8 μm. All implants were textured by acid etching in a 48% sulfuric acid bath at 60 °C while agitating with a stir bar for 30 min. The implants were rinsed in deionized water, cleaned in deionized water for 10 min under ultrasonic agitation, dehydrated in a 70% ethanol solution, and allowed to air dry before packaging for sterilization by autoclave. This procedure is similar to what has been done previously in this model.7
2.5. Statistical analysis Treatment differences in the outcome measure were evaluated by unpaired Student's t-test using a statistical analysis program (SigmaPlot v11.0, Systat Software, San Jose, CA). A significance level of 0.05 was used for detecting treatment effects. A power analysis prior to the study had determined that a sample size of 11 animals per group would be required to detect a 42% change in the pushout fixation strength for a power of 0.80 and significance level of 0.05 assuming a standard deviation of 30% of the control mean.
2.2. Surgical implantation and treatment delivery Under isoflurane anesthesia, a 2 cm longitudinal incision was made on the anteromedial aspect of the rat's knee and the patella was reflected laterally to expose the intercondylar notch of the femur. An 18gauge needle was inserted in retrograde fashion into the medullary canal of the femur, which was then reamed with a 1 mm hand-drill bit to the level of the lesser trochanter. 0.1 mL of normal saline or 0.1 ml of minocycline microspheres (1 mg/ml; OraPharma) was injected directly into the medullary cavity under direct visualization followed by immediate implantation of the titanium alloy implant. The implant was seated flush with the articular surface of the distal femur. The joint capsule was closed with absorbable suture and skin incisions closed with wound clips. The procedure was repeated for the contralateral femur. Seven days post-operatively the wound clips were removed and radiographs of both femurs were taken to ensure proper placement of the implants. The animals were allowed to heal for four-weeks, at which time they were euthanized and femurs were isolated for biomechanical testing and μCT. Following euthanasia, femurs were carefully removed and denuded to bone. The left femurs were wrapped in 0.9% salinesolution-soaked gauze and frozen at −20 °C until biomechanical testing. The right femurs were fixed in neutral buffered formalin for 48 h and transferred to 70% ethanol in preparation for μCT analysis.
3. Results Rats were 24-weeks old at the onset of the study. Mean body weights of the groups did not differ and were the following: control (392.3 ± 9.5 g) and minocycline (393.3 ± 7.9 g). 3.1. Biomechanical testing Pushout testing to failure revealed that the mean force required to pushout minocycline (122.5 ± 39.1 N) treated implants was 26% greater than the control (96.9 ± 26.1 N). However, this difference did not reach statistical significance (p = 0.098). Similarly, the energy to maximum load was slightly greater for minocycline treated implants. However, there was no significant difference in this measure between the groups. The mean stiffness was similar in both groups (p = 0.923) (Fig. 1). 3.2. μCT analysis μCT analysis determined the mean peri-implant bone volume fraction (BV/TV) of the minocycline (36.7 ± 10.8) trended to higher than the control group (30.6 ± 7.5); however, the difference was not significantly different (p = 0.182) (Fig. 2).
2.3. Biomechanical testing Prior to testing, each femur was thawed to room temperature. Once thawed, femurs were prepared by exposing the proximal end of the implant via careful sharp excision of the proximal femur until the implant was identified. The distal end of the femur was potted in a polymethylmethacrylate dental cement (Ortho Jet BCA, Lang Dental, Wheeling, IL) and the implant was subjected to a pushout test using a material testing system (8500 Plus, Instron Corp., Norwood, MA). The long axis of the implant was aligned with the axis of push-out loading and loaded at 2 mm/min until failure of the bone-implant interface was reached. Maximum push-out load (N), energy to failure (mJ), and bone-
4. Discussion The results of the present study suggest that local administration of slow release minocycline did not adversely affect osseointegration, and instead resulted in a trend toward enhanced osseointegration, suggesting that local delivery of minocycline can be used safely as a periimplant adjuvant for infection prophylaxis or the treatment of prosthetic joint infection (PJI) without compromising osseointegration. This 214
Journal of Orthopaedics 20 (2020) 213–216
J.A. Shapiro, et al.
Fig. 1. Box plots of results from push-out testing. Dashed lines represent the mean, solid lines represent the median, and tick marks represent the 5%/95% confidence intervals.
osteolysis and infection are chronic issues in arthroplasty that cannot be addressed fully during the 4-week immediate postoperative period; however, this study does address acute osseointegration failure. Furthermore, the routine use of local antibiotics for osseointegration enhancement or infection prophylaxis is controversial and may increase the prevalence of resistant organisms. Analysis in an early infection model is now warranted to investigate the combined antimicrobial and osseointegration utility of locally delivered minocycline microspheres to orthopaedic implants, as this study supports that its use would not be harmful. 5. Conclusion Peri-implant application of minocycline did not have a deleterious effect on implant fixation strength or peri-implant bone volume in our murine model, supporting that minocycline microspheres could be applied to an intramedullary implant without compromising osseointegration.
Fig. 2. Box plot of peri-implant bone volume fraction of the metaphyseal cancellous bone of the proximal femur.
is not the first time that an antibiotic has been studied for osseointegration; however, it is the first study of its kind to locally administer minocycline with a slow release delivery system that is already available for commercial use in periodontal practice. The initial dosing was designed to produce an intramedullary concentration of minocycline of 10 μg/ml – a level previously shown to not adversely influence osteoblast cell number or alkaline phosphatase activity in vitro10 – with a subsequent controlled release over the duration of the study period. While the study cannot suggest enhancement in osseointegration, it is possible that increasing or decreasing the dose may have enhanced or impeded osseointegration. Applying agents locally to enhance osseointegration is not new. Incorporating porosity in metallic implants has been shown to enhance osseointegration via bony ingrowth and can create a modulus similar to cancellous bone resulting in excellent biocompatibility.11 Coating of implants with calcium-phosphate analogues and bisphosphonates may reduce aseptic loosening.12 Furthermore, implant coating with antimicrobials covalently tethered to implants may reduce septic loosening. Erythromycin has been shown to reduce osteolysis via inhibition of inflammatory pathways and is routinely a major constituent of successful antibiotic impregnated cements.13 Prior studies have shown that local delivery of erythromycin via coating to Peri-Apatite-coated titanium implants may reduce osteolysis.14 Although controversial, local delivery of antibiotics, has been shown to reduce the rate of PJI.13 The addition of minocycline to the implant or intramedullary space would expand antimicrobial coverage to include methicillin-resistant Staphylococcus aureus which is a major pathogen in acute arthroplasty infections,15 while producing an anti-inflammatory state that at least in this study trended toward statistically significant enhanced osseointegration. There are limitations to this study. Significance was not met despite a trend in a positive difference. However, an a priori power analysis suggested adequate sample size to see a difference. Additionally,
Author contributions JAS: data analysis/interpretation, drafting the manuscript, and editing the manuscript. SA: experimental design, data collection, and editing the manuscript. CPL: experimental design, data collection, and editing the manuscript. GBM: implementation of study methods, data collection, drafting the manuscript, and editing the manuscript. LED: experimental design, senior advisor, and editing the manuscript. PSW: experimental design, implementation of study methods, data analysis/interpretation, and editing the manuscript. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Funding for this study was provided by the Aileen Stock Research Fund, and μCT imaging service was provided by the Small Animal Imaging Facility at the UNC Biomedical Imaging Research Center, which is supported in part by an NCI cancer core grant, P30-CA01608640. References 1. Drees P, Eckardt A, Gay RE, Gay S, Huber LC. Mechanisms of disease: molecular insights into aseptic loosening of orthopedic implants. Nat Clin Pract Rheumatol.
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