Publications by authors named "Stefanie M Shiels"

17 Publications

  • Page 1 of 1

Settable Polymeric Autograft Extenders in a Rabbit Radius Model of Bone Formation.

Materials (Basel) 2021 Jul 15;14(14). Epub 2021 Jul 15.

Department of Biomedical Engineering, Vanderbilt University, 2201 West End Ave, Nashville, TN 37235, USA.

Autograft (AG) is the gold standard for bone grafts, but limited quantities and patient morbidity are associated with its use. AG extenders have been proposed to minimize the volume of AG while maintaining the osteoinductive properties of the implant. In this study, poly(ester urethane) (PEUR) and poly(thioketal urethane) (PTKUR) AG extenders were implanted in a 20-mm rabbit radius defect model to evaluate new bone formation and graft remodeling. Outcomes including µCT and histomorphometry were measured at 12 weeks and compared to an AG (no polymer) control. AG control examples exhibited new bone formation, but inconsistent healing was observed. The implanted AG control was resorbed by 12 weeks, while AG extenders maintained implanted AG throughout the study. Bone growth from the defect interfaces was observed in both AG extenders, but residual polymer inhibited cellular infiltration and subsequent bone formation within the center of the implant. PEUR-AG extenders degraded more rapidly than PTKUR-AG extenders. These observations demonstrated that AG extenders supported new bone formation and that polymer composition did not have an effect on overall bone formation. Furthermore, the results indicated that early cellular infiltration is necessary for harnessing the osteoinductive capabilities of AG.
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http://dx.doi.org/10.3390/ma14143960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305944PMC
July 2021

Effects of nanocrystalline hydroxyapatite concentration and skeletal site on bone and cartilage formation in rats.

Acta Biomater 2021 Aug 12;130:485-496. Epub 2021 Jun 12.

Department of Biomedical Engineering, Vanderbilt University, 2201 West End Ave, Nashville TN 37235, United States; Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2201 West End Ave, Nashville TN 37235, United States; Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, 1211 Medical Center Dr., Nashville, TN 37212, United States; Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, 1211 Medical Center Dr., Nashville, TN 37217, United States. Electronic address:

Most fractures heal by a combination of endochondral and intramembranous ossification dependent upon strain and vascularity at the fracture site. Many biomaterials-based bone regeneration strategies rely on the use of calcium phosphates such as nano-crystalline hydroxyapatite (nHA) to create bone-like scaffolds. In this study, nHA was dispersed in reactive polymers to form composite scaffolds that were evaluated both in vitro and in vivo. Matrix assays, immunofluorescent staining, and Western blots demonstrated that nHA influenced mineralization and subsequent osteogenesis in a dose-dependent manner in vitro. Furthermore, nHA dispersed in polymeric composites promoted osteogenesis by a similar mechanism as particulated nHA. Scaffolds were implanted into a 2-mm defect in the femoral diaphysis or metaphysis of Sprague-Dawley rats to evaluate new bone formation at 4 and 8 weeks. Two formulations were tested: a poly(thioketal urethane) scaffold without nHA (PTKUR) and a PTKUR scaffold augmented with 22 wt% nHA (22nHA). The scaffolds supported new bone formation in both anatomic sites. In the metaphysis, augmentation of scaffolds with nHA promoted an intramembranous healing response. Within the diaphysis, nHA inhibited endochondral ossification. Immunohistochemistry was performed on cryo-sections of the bone/scaffold interface in which CD146, CD31, Endomucin, CD68, and Myeloperoxidase were evaluated. No significant differences in the infiltrating cell populations were observed. These findings suggest that nHA dispersed in polymeric composites induces osteogenic differentiation of adherent endogenous cells, which has skeletal site-specific effects on fracture healing. STATEMENT OF SIGNIFICANCE: Understanding the mechanism by which synthetic scaffolds promote new bone formation in preclinical models is crucial for bone regeneration applications in the clinic where complex fracture cases are seen. In this study, we found that dispersion of nHA in polymeric scaffolds promoted in vitro osteogenesis in a dose-dependent manner through activation of the PiT1 receptor and subsequent downstream Erk1/2 signaling. While augmentation of polymeric scaffolds with nHA enhanced intramembranous ossification in metaphyseal defects, it inhibited endochondral ossification in diaphyseal defects. Thus, our findings provide new insights into designing synthetic bone grafts that complement the skeletal site-specific fracture healing response.
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http://dx.doi.org/10.1016/j.actbio.2021.05.056DOI Listing
August 2021

Negative pressure wound therapy does not diminish efficacy of topical antibiotic powder in a preclinical contaminated wound model.

Bone Joint Res 2021 Feb;10(2):149-155

Orthopaedic Trauma Research Department, U.S. Army Institute of Surgical Research, San Antonio, Texas, USA.

Aims: High-energy injuries can result in multiple complications, the most prevalent being infection. Vancomycin powder has been used with increasing frequency in orthopaedic trauma given its success in reducing infection following spine surgery. Additionally, large, traumatic injuries require wound coverage and management by dressings such as negative pressure wound therapy (NPWT). NPWT has been shown to decrease the ability of antibiotic cement beads to reduce infection, but its effect on antibiotic powder is not known. The goal of this study was to determine if NPWT reduces the efficacy of topically applied antibiotic powder.

Methods: Complex musculoskeletal wounds were created in goats and inoculated with a strain of modified to emit light. Six hours after contaminating the wounds, imaging, irrigation, and debridement and treatment application were performed. Animals received either vancomycin powder with a wound pouch dressing or vancomycin powder with NPWT.

Results: There were no differences in eradication of bacteria when vancomycin powder was used in combination with NPWT (4.5% of baseline) compared to vancomycin powder with a wound pouch dressing (1.7% of baseline) (p = 0.986), even though approximately 50% of the vancomycin was recovered in the NPWT exudate canister.

Conclusion: The antimicrobial efficacy of the vancomycin powder was not diminished by the application of NPWT. These topical and locally applied therapies are potentially effective tools that can provide quick, simple treatments to prevent infection while providing coverage. By reducing the occurrence of infection, the recovery is shortened, leading to an overall improvement in quality of life. Cite this article:  2021;10(2):149-155.
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http://dx.doi.org/10.1302/2046-3758.102.BJR-2020-0171.R1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937412PMC
February 2021

Poly(Thioketal Urethane) Autograft Extenders in an Intertransverse Process Model of Bone Formation.

Tissue Eng Part A 2019 07 9;25(13-14):949-963. Epub 2019 Jan 9.

1Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee.

Impact Statement: The development of autograft extenders is a significant clinical need in bone tissue engineering. We report new settable poly(thioketal urethane)-based autograft extenders that have bone-like mechanical properties and handling properties comparable to calcium phosphate bone cements. These settable autograft extenders remodeled to form new bone in a biologically stringent intertransverse process model of bone formation that does not heal when treated with calcium phosphate bone void fillers or cements alone. This is the first study to report settable autograft extenders with bone-like strength and handling properties comparable to ceramic bone cements, which have the potential to improve treatment of bone fractures and other orthopedic conditions.
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http://dx.doi.org/10.1089/ten.TEA.2018.0223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648232PMC
July 2019

Topical rifampin powder for orthopaedic trauma part II: Topical rifampin allows for spontaneous bone healing in sterile and contaminated wounds.

J Orthop Res 2018 12 29;36(12):3142-3150. Epub 2018 Oct 29.

U.S. Army Institute of Surgical Research, 3698 Chambers Pass, Fort Sam Houston, Texas, 78234.

Infectious complications can reduce fracture healing rate. Broad spectrum antibiotics are commonly administered to prevent and treat musculoskeletal infections. Local antibiotics are applied to the wound site to increase therapeutic concentrations without increasing systemic toxicity, however, may hinder local tissue recovery. Rifampin has been shown to eradicate mature Staphylococcal biofilms and its use proven for treating musculoskeletal infections. In this study, a spontaneously healing defect model in a rat was used to investigate the impact rifampin powder has on endogenous bone healing in both a sterile and contaminated wound. No significant differences were identified in bone volume fraction via microcomputed tomography, radiological scoring, or histology between an empty defect and animals that received vancomycin or rifampin powder in a sterile wound. When applied to a contaminated musculoskeletal wound, the rifampin powder had significantly greater bone formation compared to the control, as measured by microcomputed tomography, plain radiology, and histology. In addition, the animals treated with rifampin powder had reduced bacteria, reduced white blood cell count and reduced number of clinical indications of infection. Interestingly, while the vancomycin group still displayed signs of infection via quantitative microbiology, plain radiology, and histology, there was significant bone formation within the defect and reduction of systemic signs of infection. We demonstrated that the use of rifampin powder allows bone to heal in both a sterile and contaminated model of musculoskeletal infection. To our knowledge, this is the first time the direct impact of local antibiotics on bone healing has been investigated. Published 2018. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res 36:3142-3150, 2018.
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http://dx.doi.org/10.1002/jor.24155DOI Listing
December 2018

Settable polymer/ceramic composite bone grafts stabilize weight-bearing tibial plateau slot defects and integrate with host bone in an ovine model.

Biomaterials 2018 10 26;179:29-45. Epub 2018 Jun 26.

Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, 37235, USA; Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, 37235, USA; Center for Bone Biology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37235, USA. Electronic address:

Bone fractures at weight-bearing sites are challenging to treat due to the difficulty in maintaining articular congruency. An ideal biomaterial for fracture repair near articulating joints sets rapidly after implantation, stabilizes the fracture with minimal rigid implants, stimulates new bone formation, and remodels at a rate that maintains osseous integrity. Consequently, the design of biomaterials that mechanically stabilize fractures while remodeling to form new bone is an unmet challenge in bone tissue engineering. In this study, we investigated remodeling of resorbable bone cements in a stringent model of mechanically loaded tibial plateau defects in sheep. Nanocrystalline hydroxyapatite-poly(ester urethane) (nHA-PEUR) hybrid polymers were augmented with either ceramic granules (85% β-tricalcium phosphate/15% hydroxyapatite, CG) or a blend of CG and bioactive glass (BG) particles to form a settable bone cement. The initial compressive strength and fatigue properties of the cements were comparable to those of non-resorbable poly(methyl methacrylate) bone cement. In animals that tolerated the initial few weeks of early weight-bearing, CG/nHA-PEUR cements mechanically stabilized the tibial plateau defects and remodeled to form new bone at 16 weeks. In contrast, cements incorporating BG particles resorbed with fibrous tissue filling the defect. Furthermore, CG/nHA-PEUR cements remodeled significantly faster at the full weight-bearing tibial plateau site compared to the mechanically protected femoral condyle site in the same animal. These findings are the first to report a settable bone cement that remodels to form new bone while providing mechanical stability in a stringent large animal model of weight-bearing bone defects near an articulating joint.
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http://dx.doi.org/10.1016/j.biomaterials.2018.06.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065109PMC
October 2018

Topical rifampin powder for orthopedic trauma part I: Rifampin powder reduces recalcitrant infection in a delayed treatment musculoskeletal trauma model.

J Orthop Res 2018 12 5;36(12):3136-3141. Epub 2018 Oct 5.

US Army Institute of Surgical Research, 3698 Chambers Pass, Fort Sam Houston 78234, Texas.

Open fractures become infected despite meticulous debridement and care. Locally applied antibiotics, commonly embedded in polymethylmethacrylate, deliver high doses of drug directly to the fracture site. Direct application of antibiotic powder, which is being applied prophylactically in spine surgery, is a recent interest in the trauma sector, where bacterial biofilms are more prevalent. Traditional antibiotics, such as vancomycin, are poor performers against bacterial biofilms thus are ineffective in delayed treatment. Rifampin is an effective eradicator of Staphylococcal biofilms. Here, a rat model of musculoskeletal trauma was used to evaluate the utility of locally applied rifampin powder for reducing established orthopedic Staphylococcal infections in a delayed treatment scenario that previously indicated the limited use of local vancomycin. By applying rifampin powder directly to the contaminated segmental defect, the number of bacteria, as well as clinical indications of infection, were significantly reduced compared to vancomycin and daptomycin. Considering the Infectious Disease Society of America's recommendation to use rifampin in combination with another antibiotic to reduce the onset of rifampin resistance, rifampin powder was also applied in combination with vancomycin or daptomycin with insignificant changes in eradication performance. No indications of rifampin resistance were identified. Clinical Significance: The use of locally applied rifampin is a promising therapy for mature and tolerant musculoskeletal infections. Published 2018. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res 36:3136-3141, 2018.
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http://dx.doi.org/10.1002/jor.24055DOI Listing
December 2018

Local control of polymicrobial infections via a dual antibiotic delivery system.

J Orthop Surg Res 2018 Mar 15;13(1):53. Epub 2018 Mar 15.

United States Army Institute of Surgical Research, 3855 Roger Brooke Drive, Fort Sam Houston, San Antonio, TX, 78234, USA.

Background: Contaminated traumatic open orthopedic wounds are frequently complicated by polymicrobial contamination and infection. In high-risk wounds, the standard of care comprises debridement and irrigation combined with antibiotics which can be applied directly or combined with systemic antibiotics. Recently, bioabsorbable chitosan sponges have been shown to be an effective single-agent delivery device for local antibiotics with and without negative pressure wound therapy (NPWT). Severely contaminated orthopedic wounds, however, are often complicated by polymicrobial infections, necessitating multiple antibiotic agents. As such, the purpose of this study was to determine if a chitosan sponge would provide a suitable delivery vehicle for multiple antibiotics for the treatment of a polymicrobial infection in a large animal polytraumatic extremity wound model.

Methods: A complex polytraumatic extremity wound was created in 11 adult male Boer goats. Each wound was contaminated with a bioluminescent strain of S. aureus (1 ml of 10 colony forming units/ml) and of P. aeruginosa (1 ml of 10 CFU/ml) which are genetically engineered to allow quantification with a photon-counting camera. Six hours following initial wound creation and contamination, wounds were debrided and irrigated with low-pressure normal saline. The animals were randomized into one of two treatments: wet-to-dry dressings alone or a commercially available chitosan sponge loaded with 1 g vancomycin and 1.2 g of tobramycin. Each animal was then recovered and reimaged 48 h later for total bacteria content; tissue samples were taken from the wound bed to determine relative bacterial colonization.

Results: All animals in the chitosan sponge group saw significant reductions in overall bacterial load of S. aureus and P. aeruginosa (p = 0.001). The bioluminescence was also significantly reduced compared to the wet-to-dry dressing group (p = 0.0001). Furthermore, whereas the antibiotic sponge group displayed near complete eradication of bacteria, the wounds treated with the wet-to-dry dressings alone displayed a significant 2-log increase in total bacteria at 48 h p = 0.0001). S. aureus was the predominant species found in the wounds, comprising 95 and 99% of all bacteria found in the chitosan sponge and wet-to-dry, respectively.

Conclusion: Dual antimicrobial therapy loaded in a chitosan sponge is an effective way to reduce polymicrobial infections traumatic extremity wound.
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http://dx.doi.org/10.1186/s13018-018-0760-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856197PMC
March 2018

Determining potential of PMMA as a depot for rifampin to treat recalcitrant orthopaedic infections.

Injury 2017 Oct 16;48(10):2095-2100. Epub 2017 Aug 16.

Extremity Trauma and Regenerative Medicine Task Area, US Army Institute of Surgical Research, JBSA-Fort Sam Houston, TX, United States.

Background: Open fractures are often complicated by infection. In cases of severe soft tissue and vascular injury, systemic antibiotics may be ineffective due to their inability to reach and provide direct antimicrobial activity to the zone of injury. High antibiotic concentrations within the wound can be achieved with reduced systemic toxicity by using local antibiotic delivery. As bacteria associated with musculoskeletal injuries frequently form biofilms, antibiotic selection is important. Herein, the use of rifampin, an antibiotic with activity against biofilms, delivered via polymethylmethacrylate (PMMA) beads is evaluated for use in a traumatic musculoskeletal wound model.

Methods: PMMA beads loaded with rifampin, or combinations of rifampin and vancomycin, were prepared and evaluated for time to curing, drug release kinetics in vitro, and infection prevention in vivo using a well-established rat model of musculoskeletal infection. A segmental bone defect was created and contaminated with methicillin susceptible Staphylococcus aureus (UAMS-1). Wounds were debrided, irrigated, and treated with PMMA beads, containing rifampin or combinations of rifampin plus vancomycin, following a 6-h (early) or 24-h (delayed) treatment. After 14days, tissue, implants, and beads were removed for bacterial quantification and assessed for rifampin resistance.

Results: There was a direct association between loaded concentration and release kinetics of the rifampin and vancomycin from PMMA beads. Higher rifampin concentrations delayed PMMA curing times. The addition of vancomycin to PMMA resulted in more rapid release of rifampin from beads. However, the highest concentration of rifampin loaded PMMA beads (10% wt/wt) was the only treatment to significantly reduce bacterial counts. No rifampin resistance was observed.

Conclusion: Although higher concentrations of rifampin resulted in significant reductions of bacteria, these levels extended PMMA curing times and transformed PMMA material characteristics. While these characteristics make the material unsuitable for weight-bearing applications, such as total joint arthroplasty, the use of rifampin-loaded PMMA beads may be an effective intervention in a contaminated traumatic extremity wound due to its ability to eradicate biofilms.
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http://dx.doi.org/10.1016/j.injury.2017.08.021DOI Listing
October 2017

Injectable and compression-resistant low-viscosity polymer/ceramic composite carriers for rhBMP-2 in a rabbit model of posterolateral fusion: a pilot study.

J Orthop Surg Res 2017 Jul 11;12(1):107. Epub 2017 Jul 11.

US Army Institute of Surgical Research, Fort Sam Houston, TX, USA.

Background: The challenging biological and mechanical environment of posterolateral fusion (PLF) requires a carrier that spans the transverse processes and resists the compressive forces of the posterior musculature. The less traumatic posterolateral approach enabled by minimally invasive surgical techniques has prompted investigations into alternative rhBMP-2 carriers that are injectable, settable, and compression-resistant. In this pilot study, we investigated injectable low-viscosity (LV) polymer/composite bone grafts as compression-resistant carriers for rhBMP-2 in a single-level rabbit PLF model.

Methods: LV grafts were augmented with ceramic microparticles: (1) hydrolytically degradable bioactive glass (BG), or (2) cell-degradable 85% β-tricalcium phosphate/15% hydroxyapatite (CM). Material properties, such as pore size, viscosity, working time, and bulk modulus upon curing, were measured for each LV polymer/ceramic material. An in vivo model of posterolateral fusion in a rabbit was used to assess the grafts' capability to encourage spinal fusion.

Results: These materials maintained a working time between 9.6 and 10.3 min, with a final bulk modulus between 1.2 and 3.1 MPa. The LV polymer/composite bone grafts released 55% of their rhBMP-2 over a 14-day period. As assessed by manual palpation in vivo, fusion was achieved in all (n = 3) animals treated with LV/BG or LV/CM carriers incorporating 430 μg rhBMP-2/ml. Images of μCT and histological sections revealed evidence of bone fusion near the transverse processes.

Conclusion: This study highlights the potential of LV grafts as injectable and compression-resistant rhBMP-2 carriers for posterolateral spinal fusion.
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http://dx.doi.org/10.1186/s13018-017-0613-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504717PMC
July 2017

Antibiotic-loaded bone graft for reduction of surgical site infection in spinal fusion.

Spine J 2017 12 3;17(12):1917-1925. Epub 2017 Jul 3.

Extremity Trauma and Regenerative Medicine Task Area, United States Army Institute of Surgical Research, 3698 Chambers Pass, JBSA Fort Sam Houston, TX 78234, USA.

Background Context: Infections remain a leading complication associated with spinal arthrodesis, regardless of the use of prophylactic antibiotics and improved surgical techniques, with incidence of infection as high as 8.2%. Infection prolongs antibiotic usage, increases hospital time, and inevitably inflates overall treatment costs. Local antibiotics, such as vancomycin, have been used in combination with fusion materials over the past decade to decrease infection risk. An ideal graft material would serve a dual role: encouraging vertebral fusion while reducing the incidence of infection.

Purpose: The objective of this study was to thoroughly evaluate the use of a vancomycin-loaded demineralized bone matrix (vDBM) for fusion capability while reducing the incidence of surgical site infection.

Study Design: Antimicrobial efficacy and spinal fusion were evaluated using a preclinical rabbit model of posterolateral fusion.

Materials And Methods: Vancomycin-loaded demineralized bone matrix was prepared and evaluated for in vitro release kinetics and bacterial inhibition. In vivo antibacterial efficacy and fusion capability were performed using a model of posterolateral fusion in a rabbit. First, 10 New Zealand white rabbits underwent a bilateral posterolateral fusion procedure, were inoculated with Staphylococcus aureus, and were treated with either demineralized bone matrix (DBM) or vDBM. Fourteen days after the procedure, the animals were anesthetized and euthanized, and the transverse process was harvested and enumerated for bacterial quantification. Concurrently, 21 New Zealand white rabbits underwent the same procedure and were euthanized 8 weeks after surgery and were evaluated for fusion by manual palpation and radiographic scoring. In addition, two groups of six animals received the DBM or vDBM material as described, but the graft was combined with equal volumes of milled harvest iliac crest bone graft (ICBG). Eight weeks after surgery, these animals were euthanized and also evaluated for fusion by manual palpation and radiographic scoring.

Results: Vancomycin continued to be released from the vDBM over the course of 6 days while maintaining sufficient eluate concentrations to maintain a zone of inhibition similar or larger than a vancomycin control. In vivo, vDBM significantly reduced the amount of bacteria within the fusion site compared with DBM, with a 4-log decrease in bacterial bioburden. The use of vDBM, however,showed a decrease in the fusion rate compared with DBM when used in a sterile wound. In a S. aureus-contaminated wound, both the DBM and the vDBM showed decreased fusion rates.Considering DBM materials were most commonly used as autograft extenders, additional animals received either DBM plus ICBG in a sterile wound or vDBM plus ICBG in a contaminated wound. Both groups had similar fusion rates and similar fusion volumes after 8 weeks in vivo.

Conclusions: Whereas vDBM reduced the overall bioburden within a contaminated surgical site of posterolateral fusion, the addition of the vancomycin to the DBM reduced the fusion capability of the DBM graft. The addition of ICBG to vDBM restored the fusion capability of the graft while reducing the overall infection.
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http://dx.doi.org/10.1016/j.spinee.2017.06.039DOI Listing
December 2017

Time-Dependent Effectiveness of Locally Applied Vancomycin Powder in a Contaminated Traumatic Orthopaedic Wound Model.

J Orthop Trauma 2016 Oct;30(10):531-7

*United States Army Institute of Surgical Research, TX; and †Department of Orthopaedics, San Antonio Military Medical Center, TX.

Objectives: To evaluate the effectiveness of locally applied vancomycin powder at different times postinfection in a contaminated traumatic animal model.

Methods: This study used an established segmental defect rat femur model contaminated with Staphylococcus aureus UAMS-1 followed by treatment at 6 or 24 hours postinfection. Three treatments were evaluated: debridement and irrigation alone (control group) or in combination with either vancomycin powder or vancomycin-impregnated poly(methyl methacrylate) beads. Serum vancomycin levels were determined at scheduled time points over 14 days; bone, surrounding muscle, and implants were harvested for bacterial and inflammatory analyses.

Results: Locally applied vancomycin powder and impregnated beads significantly reduced bacteria both within the bone and implant when treatment was performed at 6 hours. Delaying treatment to 24 hours significantly reduced the therapeutic efficacy of locally applied vancomycin of both groups. Serum vancomycin levels were detectable in all animals treated with vancomycin powder at 24 hours, but absorption was negligible from beads. At 14 days, vancomycin was detectable in the surrounding musculature of all animals and in serum of 20% of animals treated with vancomycin powder.

Conclusions: This study suggests that vancomycin powder is a promising adjunctive therapy for preventing infection in traumatic wounds when treatment is performed early. This time-dependent effectiveness of vancomycin powder is similar to that observed with systemic and other local delivery adjuncts, which is likely attributable to biofilm formation after contamination, conferring intrinsic recalcitrance to antimicrobials.
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http://dx.doi.org/10.1097/BOT.0000000000000617DOI Listing
October 2016

Development of a hematogenous implant-related infection in a rat model.

BMC Musculoskelet Disord 2015 Sep 14;16:255. Epub 2015 Sep 14.

Extremity Trauma and Regenerative Medicine Task Area, US Army Institute of Surgical Research, JBSA-Fort Sam Houston, TX, 78234, USA.

Background: Implant-related osteomyelitis is a major complication that requires immediate treatment, often involving removal of the implant, prolonging patient recovery and inflating expenses. Current research involving interventions to diminish the prevalence of such measures include investigating prophylactic and therapeutic remedies. A proper and accurate animal model is needed to thoroughly investigate such treatments. The scope of this project was to develop an animal model in which a consistent and measurable infection can be formed on an orthopedic implant when bacteria is introduced via a hematogenous source.

Methods: Titanium Kirschner-wires were implanted into the intramedullary canals of both femurs. Staphylococcus aureus, ranging from10(4) to 10(9) colony forming units, was injected into a tail vessel. After a designated time (3, 7, 14, or 42 days) the femurs were harvested and bacterial numbers determined for both the femur and the implanted K-wire. In addition, histology and micro-computed tomography were used as subjective tools to further characterize the infection.

Results: Consistent infection, that is infection of ≥75% of the femurs, wasn't achieved until 10(7) CFU S. aureus was injected. At 10(7) CFU, the femurs contained 4.6x10(6) CFU/g bone tissue at day 3 and 4.8×10(8) CFU/g bone tissue by day 14. The wire showed comparable contamination with 4.8×10(4) CFU/mm(2) at day 3 and 3.7×10(5)/mm(2) by day 14. After 42 days, the bacteria number decreased but was still occupying at 1.9×10(5) CFU/g bone tissue. There were morphological changes to the bone as well. At day 42, there were signs of osteonecrosis and active bone formation when compared to control animals that received a K-wire but were inoculated with saline.

Conclusions: A model for hematogenous osteomyelitis, a common complication associated with implants, has been introduced. A reproducible, preclinical model is essential to evaluate future methods used to mitigate blood-borne bacteria hardware and bone infections.
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http://dx.doi.org/10.1186/s12891-015-0699-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4570216PMC
September 2015

Alternatives to autograft evaluated in a rabbit segmental bone defect.

Int Orthop 2016 Jan 9;40(1):197-203. Epub 2015 Jul 9.

Extremity Trauma and Regenerative Medicine, United States Army Institute of Surgical Research, 3698 Chambers Pass BLDG 3611, JBSA Fort Sam Houston, TX, 78234, USA.

Purpose: This study was designed to identify strategies for treating bone defects that can be completed on the day of surgery.

Methods: Forty New Zealand white rabbits with unilateral rabbit radius segmental defects (15 mm) were treated with commercially available scaffolds containing either demineralised bone matrix (DBM) or a collagen/beta-tricalcium phosphate composite (Col:β-TCP); each scaffold was combined with either bone marrow aspirate (BMA) or concentrated BMA (cBMA). Bone regeneration was assessed through radiographic and histological analyses.

Results: The concentration of nucleated cells, colony-forming unit-fibroblasts and platelets were increased and haematocrit concentration decreased in cBMA as compared to BMA (p < 0.05). Radiographic analyses of bone formation and defect bridging demonstrated significantly greater bone regeneration in the defects treated with DBM grafts as compared to Col:β-TCP grafts. The healing of bones treated with Col:β-TCP was improved when augmented with cBMA.

Conclusions: Scaffolds containing either DBM or Col:β-TCP with BMA or cBMA are effective same-day strategies available to clinicians for the treatment of bone defects; the latter scaffold may be more effective if combined with cBMA.
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http://dx.doi.org/10.1007/s00264-015-2824-5DOI Listing
January 2016

Rifamycin Derivatives Are Effective Against Staphylococcal Biofilms In Vitro and Elutable From PMMA.

Clin Orthop Relat Res 2015 Sep;473(9):2874-84

Extremity Trauma & Regenerative Medicine Task Area, US Army Institute of Surgical Research, 3698 Chambers Pass, JBSA, Fort Sam Houston, TX, 78234, USA.

Background: Local antimicrobial delivery through polymethylmethacrylate beads (PMMA), commonly vancomycin, is used for the treatment of contaminated open fractures but has limited activity against Staphylococcus aureus biofilms, which occur commonly in such fractures. Rifamycins have activity against biofilms and are an effective treatment for osteoarticular infections involving staphylococcal biofilms, but there are limited studies evaluating the activity of rifamycin derivatives, other than rifampin, against biofilms of S. aureus and evaluating incorporation of these drugs into PMMA for treatment of contaminated open fractures.

Questions/purposes: (1) Are rifamycin derivatives effective against established biofilms of clinical isolates of S. aureus? (2) Can PMMA be used as a carrier for rifamycin derivatives?

Methods: Biofilms were developed and evaluated for susceptibility to a panel of antimicrobials in vitro using the minimum biofilm eradication concentration high-throughput model. Susceptibility was assessed by measuring bacterial recovery at 6 and 24 hours after antimicrobial treatment. Activity of rifamycin derivatives against intracellular bacteria was also evaluated using a gentamicin protection assay. Evaluation of PMMA as a carrier for rifampin and rifamycin derivatives was determined by assessing the curing time subsequent to loading of rifamycins and characterizing the release kinetics of rifamycins at daily intervals for 14 days from PMMA by performing bioassays.

Results: Rifamycin derivatives between 1 and 8 µg/mL reduced bacteria within biofilms 5- to 9-logs and prevented bacterial recovery up to 24 hours post-treatment, indicating near to complete eradication of biofilms. Rifamycin derivatives at 32 µg/mL had activity against intracellular staphylococci, significantly reducing the number of internalized bacteria with limited effects on osteoblast viability. Rifampin was the only rifamycin observed to have a suitable release profile from PMMA, releasing 49% of the total antibiotic and maintaining a sustained released profile up to 14 days at a mean 28 ± 6 μg/mL.

Conclusions: Rifampin can be incorporated into PMMA and eluted at concentrations effective against biofilms and intracellular staphylococci.

Clinical Relevance: Our in vitro findings suggest that local delivery of rifampin may be an effective strategy for the prevention and/or treatment of open fractures where S. aureus biofilms might develop. Clinical studies are needed to characterize what role this approach might have in the prevention and treatment of infections involving biofilms.
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http://dx.doi.org/10.1007/s11999-015-4300-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523531PMC
September 2015

Influence of substrate curvature on osteoblast orientation and extracellular matrix deposition.

J Biol Eng 2013 Oct 3;7(1):23. Epub 2013 Oct 3.

Department of Biomedical Engineering, University of Texas, San Antonio, TX, USA.

Background: The effects of microchannel diameter in hydroxyapatite (HAp) substrates on osteoblast behavior were investigated in this study. Microchannels of 100, 250 and 500 μm diameter were created on hydroxyapatite disks. The changes in osteoblast precursor growth, differentiation, extra cellular matrix (ECM) secretion and cell attachment/orientation were investigated as a function of microchannel diameter.

Results: Curvature did not impact cellular differentiation, however organized cellular orientation was achieved within the 100 and 250 μm microchannels (mc) after 6 days compared to the 12 days it took for the 500mc group, while the flat substrate remained disorganized. Moreover, the 100, 250 and 500mc groups expressed a specific shift in orientation of 17.45°, 9.05°, and 22.86° respectively in 24 days. The secreted/mineralized ECM showed the 100 and 250mc groups to have higher modulus (E) and hardness (h) (E = 42.6GPa; h = 1.6GPa) than human bone (E = 13.4-25.7GPa; h = 0.47-0.74GPa), which was significantly greater than the 500mc and control groups (p < 0.05). It was determined that substrate curvature affects the cell orientation, the time required for initial response, and the shift in orientation with time.

Conclusions: These findings demonstrate the ability of osteoblasts to organize and mineralize differentially in microchannels similar to those found in the osteons of compact bone. These investigations could lead to the development of osteon-like scaffolds to support the regeneration of organized bone.
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http://dx.doi.org/10.1186/1754-1611-7-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3851034PMC
October 2013

BMP-2 tethered hydroxyapatite for bone tissue regeneration: coating chemistry and osteoblast attachment.

J Biomed Mater Res A 2012 Nov 20;100(11):3117-23. Epub 2012 Jul 20.

Department of Biomedical Engineering, The University of Texas at San Antonio, San Antonio, Texas, USA.

The goal of this study was to determine the effectiveness of using polyethyleneimine (PEI) and a polyethylene glycol (PEG) tether to bind human recombinant bone morphogenetic protein-2 (rhBMP-2) to hydroxyapatite (HAp) to enhance rhBMP-2 loading, alter its release properties, and enhance cellular interaction with the material. By using a branched PEI that was derived to express free thiols, rhBMP-2 was coated onto dense HAp surfaces at ~43 ng/cm(2). Using this novel attachment methodology, it was observed that the PEI-SH coating did not change the morphology of the HAp surfaces and that the amount of rhBMP-2 loaded was comparable to a direct adsorption method. In addition, it was also observed that the PEI and PEG tether significantly retained the rhBMP-2 to the HAp surface, inhibiting the burst release effect. Using human fetal osteoblast cells, the PEI- and PEG-tethered BMP-2 was also observed to increase cellular attachment by 10-fold when compared with uncoated HAp and adsorbed rhBMP-2. It was concluded from this study that PEI and PEG tether significantly reduce the initial burst release effect of rhBMP-2. It was also concluded that the rhBMP-2 conjugation to PEI and PEG tether promoted an increase in cellular attachment to the HAp surface.
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http://dx.doi.org/10.1002/jbm.a.34241DOI Listing
November 2012
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