We have shown previously that creatine kinase (CK) activity is required for normal development an... more We have shown previously that creatine kinase (CK) activity is required for normal development and mineralization of chicken growth cartilage and that expression of the cytosolic isoforms of CK is related to the biosynthetic and energy status of the chondrocyte. In this study, we have characterized changes in isoenzyme activity and mRNA levels of CK (muscle-specific CK, M-CK; brain-type CK, B-CK; and mitochondrial CK subunits, MiaCK and MibCK) in the growth plate in situ and in chondrocyte culture systems that model the development/maturation program of the cartilage. The in vitro culture systems analyzed were as follows: tibial chondrocytes, which undergo hypertrophy; embryonic cephalic and caudal sternal chondrocytes, which differ from each other in their mineralization response to retinoic acid; and long-term micromass cultures of embryonic limb mesenchymal cells, which recapitulate the chondrocyte differentiation program. In all systems analyzed, B-CK was found to be the predominant isoform. In the growth plate, B-CK expression was highest in the most calcified regions, and M-CK was less abundant than B-CK in all regions of the growth plate. In tibial chondrocytes, an increase in B-CK expression was seen when the cells became hypertrophic. Expression of B-CK increased slightly over 15 days in mineralizing, retinoic acid-treated cephalic chondrocytes, but it decreased in nonmineralizing caudal chondrocytes, while there was little expression of M-CK. Interestingly, in limb mesenchyme cultures, significant M-CK expression was detected during chondrogenesis (days 2-7), whereas hypertrophic cells expressed only B-CK. Finally, expression of MiaCK and MibCK was low both in situ and in vitro. These observations suggest that the CK genes are differentially regulated during cartilage development and maturation and that an increase in CK expression is important in initiating chondrocyte maturation.
We have previously shown that thyroid stimulating hormone-β (TSHβ) mRNA levels are modulated by v... more We have previously shown that thyroid stimulating hormone-β (TSHβ) mRNA levels are modulated by vitamin A status in vivo and using transient transfection, that suppression of rat TSHβ gene promoter activity by all-trans retinoic acid (RA) requires RA receptor (RAR) and retinoid X receptor (RXR). In this paper we have used deletion analysis to delineate the sequences of the rTSHβ
Many surgical procedures require the placement of an inert or tissue-derived implant deep within ... more Many surgical procedures require the placement of an inert or tissue-derived implant deep within the body cavity. While the majority of these implants do not become colonized by bacteria, a small percentage develops a biofilm layer that harbors invasive microorganisms. In orthopaedic surgery, unresolved periprosthetic infections can lead to implant loosening, arthrodeses, amputations and sometimes death. The focus of this review is to describe development of an implant in which an antibiotic tethered to the metal surface is used to prevent bacterial colonization and biofilm formation. Building on well-established chemical syntheses, studies show that antibiotics can be linked to titanium through a self-assembled monolayer of siloxy amines. The stable metal-antibiotic construct resists bacterial colonization and biofilm formation while remaining amenable to osteoblastic cell adhesion and maturation. In an animal model, the antibiotic modified implant resists challenges by bacteria that are commonly present in periprosthetic infections. While the long-term efficacy and stability is still to be established, ongoing studies support the view that this novel type of bioactive surface has a real potential to mitigate or prevent the devastating consequences of orthopaedic infection.
Retinoic acid (RA) is important for normal mammalian development and growth. Ornithine decarboxyl... more Retinoic acid (RA) is important for normal mammalian development and growth. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the biosynthesis of the polyamines, and we have previously shown that ODC mRNA levels are suppressed by RA in human skin cells. Using HeLa cells, we now show that treatment with 0.5 fM RA for 24 h suppresses endogenous ODC mRNA levels and the expression of a transfected ODC/chloramphenicol acetyltransferase plasmid (Kpn-ODCCAT), containing sequences from-1450 to + 810 of the human ODC gene. Co-transfection with either the a-RA receptor (a-RAR) or a chimeric a-RA/oestrogen receptor (a-RAER) followed by treatment with the cognate hormone suppresses expression of Kpn-ODCCAT and Not-ODCCAT, which con
Journal of the American Academy of Orthopaedic Surgeons, Apr 1, 2015
ABSTRACT Orthopaedic infections are complex conditions that require immediate diagnosis and accur... more ABSTRACT Orthopaedic infections are complex conditions that require immediate diagnosis and accurate identification of the causative organisms to facilitate appropriate management. Conventional methodologies for diagnosis of these infections sometimes lack accuracy or sufficient rapidity. Molecular diagnostics is an emerging area of bench-to-bedside research in orthopaedic infections. Examples of promising molecular diagnostics include measurement of a specific biomarker in the synovial fluid, polymerase chain reaction-based detection of bacterial genes, and metabolomic determination of responses to orthopaedic infection. Copyright 2015 by the American Academy of Orthopaedic Surgeons.
Clinical Orthopaedics and Related Research, Aug 1, 2007
Results presented are not the statement or policy of the funding agencies. Each author certifies ... more Results presented are not the statement or policy of the funding agencies. Each author certifies that his or her institution has approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.
Clinical Orthopaedics and Related Research, May 15, 2019
We would like to correct errors found in our published study, “Surgical Loupes Worn by Orthopaedi... more We would like to correct errors found in our published study, “Surgical Loupes Worn by Orthopaedic Surgeons Are a Reservoir for Microorganisms”. In the Statistical Analysis section, we wrote: “Additionally, we used the Student’s t-test and the Wilcoxon signed-rank test to detect differences in categorical bacterial growth between paired samples.” Using a Student’s t-test or Wilcoxon tests is not adequate for the analysis given that these were not continuous but rather categorical variables. After a statistical consultation, we determined that the better statistical test in this setting is the Mann-Whitney U test. Given that the data does not change, the medians and 95% confidence intervals for the comparisons (before and after surgical day, before and after alcohol wipe) do not change. The statistical results of the Mann-Whitney U test are as follows: For the before and after surgical day comparison, analysis of the data sets with the Mann-Whitney U test yields a U-value of 20 and given that the critical value of U at p < 0.05 is 20, the result remains significant at p < 0.05. The z-score is 1.77604, yielding a p = 0.038. For the before and after alcohol wipe comparison, analysis of the data sets with the Mann-Whitney U test yields a U-value of 2.5 and given that the critical value of U at p < 0.05 is 5, the result remains significant at p < 0.05. The z-score is 2.40192, yielding a p = 0.02. As a result, our differences between these groups for both comparisons remain statistically different, although the p values change. Additionally, in the Discussion section, we wrote: “Lastly, we did not conduct clinical followup on the patients included in this study, and we describe a potential association of pathogen colonization with SSIs without demonstrating causation.” We intended to remove this sentence in the course of editing the paper but did not do so. The correct sentence should be: “Lastly, although particulate shedding from the loupes into the surgical field containing microorganisms could increase the risk of SSI, we did not assess SSI in the current study. Future studies should seek to evaluate the degree to which shedding of this sort might be clinically important, and if it is, to identify the best ways to mitigate it.” Finally, there was a typographical error in Table 1. Loupes of 2.5 magnification were used by 15 surgeons and those of 3.5 magnification were used by six surgeons, for a total of 21 surgeons. The authors apologize for the errors. Erratum to: Clin Orthop Relat Res DOI: 10.1097/CORR. 0000000000000651. The online version of this article can be found under DOI: 10.1097/CORR.0000000000000651.
Infection associated with inert implants is complicated by bacterial biofilm formation that rende... more Infection associated with inert implants is complicated by bacterial biofilm formation that renders the infection antibiotic insensitive. The goal of this investigation was to synthesize and characterize a vancomycin (VAN)modified bone allograft that could render the tissue inhospitable to bacterial colonization and the establishment of infection. We found that the numbers of primary amines, which could serve as anchors for chemical synthesis, increased with limited demineralization. Using these amines, we coupled two linkers and VAN to bone using Fmoc chemistry. By immunohistochemistry, VAN was abundant on the surface of the allograft; based on elution and measurement of bound antibody, this coupling yielded at least *26 ng VAN=mg bone. The coupled VAN appeared to be permanently bound to the allograft, as it showed no elution in a disk diffusion assay, and, importantly, resisted colonization by Staphylococcus aureus challenges. We suggest that this chimeric construct represents a new generation of antibiotic-modified allografts that provide antibacterial properties.
Antimicrobial Agents and Chemotherapy, Feb 1, 2011
Infection is an important medical problem associated with the use of bone allografts. To retard b... more Infection is an important medical problem associated with the use of bone allografts. To retard bacterial colonization, we have recently reported on the modification of bone allografts with the antibiotic vancomycin (VAN). In this report, we examine the ability of this antibiotic-modified allograft to resist bacterial colonization and biofilm formation. When antibiotic was coupled to the allograft, a uniform distribution of the antibiotic was apparent. Following challenges with Staphylococcus aureus for 6 h, the covalently bonded VAN decreased colonization as a function of inoculum, ranging from 0.8 to 2.0 log 10 CFU. Furthermore, the VAN-modified surface resisted biofilm formation, even in topographical niches that provide a protected environment for bacterial adhesion. Attachment of the antibiotic to the allograft surface was robust, and the bonded VAN was stable whether incubated in aqueous media or in air, maintaining levels of 75 to 100% of initial levels over 60 days. While the VAN-modified allograft inhibited the Gram-positive S. aureus colonization, in keeping with VANs spectrum of activity, the VAN-modified allograft was readily colonized by the Gram-negative Escherichia coli. Finally, initial toxicity measures indicated that the VAN-modified allograft did not influence osteoblast colonization or viability. Since the covalently tethered antibiotic is stable, is active, retains its specificity, and does not exhibit toxicity, it is concluded that this modified allograft holds great promise for decreasing bone graft-associated infections.
Introduction: The purpose of this study was to evaluate bacterial adherence to common casting mat... more Introduction: The purpose of this study was to evaluate bacterial adherence to common casting materials including plaster of Paris (plaster), fiberglass, three-dimensional (3D) printed plastic, and siliconecoated 3D printed plastic. Methods: The minimal inhibitory concentration of a phosphate-free detergent (Palmolive) needed to achieve total bacterial kill off was determined. 3D printed polylactic acid plastic samples were coated with silicone. Plaster, fiberglass, plastic, and silicone-coated plastic samples were inoculated with Staphylococcus aureus. After bacterial inoculation, scanning electron microscopy of the samples was performed to visualize bacterial adherence to the materials' surface. Using either sterile water or a 5% detergent solution, the materials were subjected to washings. Each material was run in 30 replicates: 6 without washing, 6 with sterile water for 1 minute, 6 with detergent for 1 minute, 6 with sterile water for 3 minutes, and 6 with detergent for 3 minutes. The replicates that did not undergo a washing trial represented the initial bacterial inoculation. Samples were then rinsed and sonicated in polysorbate to isolate the remaining adherent bacteria on the materials' surface. The sonicated solutions were plated, incubated, and counted for quantification of colony forming units (CFU) of bacteria. This protocol was repeated for a total of four trials. Results: During inoculation, there were significantly less bacteria that adhered to silicone-coated 3D printed plastic (58879 CFU) compared to plastic (217479 CFU), plaster (140063 CFU), and fiberglass (550546 CFU). Silicone coating showed further superiority. Silicone-coated 3D printed plastic was able to be decontaminated as demonstrated by significantly fewer remaining bacteria (9.3%) on its surface after being washed with a 5% detergent solution (1797 CFU) compared to sterile water (19321 CFU). The mean remaining bacteria on silicone-coated 3D printed plastic was significantly less than that remaining on all other materials when washed with either sterile water or a detergent solution for both durations of 1 minute and 3 minutes. Conclusions: The current study demonstrates that significantly less bacteria adhere to the surface of 3D printed plastic with silicone coating showing added protection and that this material can be decontaminated to a greater degree with washing than conventional casting materials. These results provide evidence that 3D printed casts can be washed and successfully decontaminated during a patient's period of immobilization, which is advantageous especially during an infectious crisis such as the coronavirus disease 2019 (COVID-19) pandemic.
Changes in cell shape occur during the cell cycle and influence cell proliferation and differenti... more Changes in cell shape occur during the cell cycle and influence cell proliferation and differentiation. In order to study how altered cell proliferation and cell shape are interrelated, we have studied ornithine decarboxylase (ODC) regulation in cultured normal human epidermal keratinocytes (NHEK). Cytoskeletal disruptors have been reported to modulate regulation of ODC; the products of ODC, the polyamines, influence actin polymerization rates in vitro, and polyamine auxotrophs have profoundly disrupted cytoskeletons. Therefore, altered ODC levels could be involved in signaling changes in cell shape and an intact cytoskeleton could transduce signals to regulate ODC levels. We had previously observed that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which profoundly alters cell shape, markedly suppresses ODC biosynthesis in NHEK solely at posttranscriptional/protein synthesis levels. TPA treatment caused NHEK to rapidly assume a rounded morphology that was accompanied by a change in actin organization, as determined by rhodamine-phalloidin labeling. Immunolocalization of ODC showed a perinuclear/nuclear distribution in untreated NHEK and a more diffuse pattern after TPA treatment that was apparent within 15-30 min. Changes in ODC enzyme activity are not significant until 60 min after TPA treatment. NHEK treated with cytochalasin B or D to inhibit actin polymerization exhibited a diffuse ODC localization that could be reversed by removal of the cytochalasin; inhibition of ODC by alpha-difluoromethylornithine caused a diffuse ODC localization. All treatments resulted in cytoskeletal remodeling. These data are the first evidence for a distinct subcellular localization for ODC and suggest that changes in ODC localization may be an initial step in regulation of ODC activity. Furthermore, changes in ODC activity cause an altered cytoskeleton, suggesting one means by which growth regulatory signals can be transduced to the cytoskeleton from various signaling pathways.
Bioorganic & Medicinal Chemistry Letters, May 1, 2007
Self-protecting Ti6Al4V alloy pins were prepared by covalent bonding of bis(ethylene glycol) link... more Self-protecting Ti6Al4V alloy pins were prepared by covalent bonding of bis(ethylene glycol) linkers, then vancomycin to the oxidized, aminopropylated Ti6Al4V alloy surface. Fluorescence modification-enabled estimation of yields of free amines on the metallic surface monolayer at each reaction step. The vancomycin-protected Ti6Al4V pins were not colonized by Staphylococcus aureus, even after 44 days storage in physiological buffer. These results provide a basis for testing self-protection against S. aureus colonization in animal models.
Clinical Orthopaedics and Related Research, Feb 6, 2019
Background Surgeons frequently use optical loupes to magnify the surgical field; they are typical... more Background Surgeons frequently use optical loupes to magnify the surgical field; they are typically unprotected when positioned directly over the wound, where particulate shedding containing microorganisms could potentially lead to surgical site infections (SSIs). SSIs are rare in some orthopaedic subspecialties such as hand surgery; however, in other subspecialties, for example, the spine, where surgeons often use loupes, SSIs can have devastating consequences. Questions/purposes (1) What is the degree of bacterial and fungi organism colonization of surgical loupes and storage cases? (2) Is there a difference in the degree of colonization at the beginning and the end of a surgery day? (3) Does an alcohol swab reduce bacterial colonization of surgical loupes? Methods The surgical loupes of 21 orthopaedic surgeons from a large, regional orthopaedic practice were cultured over a 3-month period and form the basis of this study. Five loupe storage cases were also cultured. In two different subgroup comparisons, the presence of microorganisms was evaluated just before the start and immediately after the end of the surgical day (n = 9) and before and 1 minute after cleaning with an alcohol swab (n = 6). A total of 36 cultures were evaluated. Surgeons who declined to participate in the study were excluded. The number of loupes selected for all of the analyses were samples of convenience and limited by surgeon availability. The degree of bacterial and fungal presence was graded using a point system: 0 = no growth; 1 = limited growth (meaning few scattered colonies); 2 = moderate growth; 3 = extensive but scattered growth; and 4 = growth consuming the entire plate. Demographic data were assessed using descriptive statistics. Additionally, the Student’s t and Wilcoxon signed-rank tests were used to detect differences in categorical bacterial growth between paired samples. A p value of 0.05 represented statistical significance. Kappa statistics of reliability were performed to evaluate interobserver agreement of microorganism growth in the culture plates. Results Bacteria were present in 19 of 21 (90%) sets of loupes. Five species of bacteria were noted. Fungi were present in 10 of 21 (48%) sets of loupes. Bacterial contamination was identified in two storage cases (40%) and fungi were present in five cases (100%). In a subset of nine loupes tested, the degree of bacterial presence had a median of 2 (range, 1-4; 95% confidence interval [CI], 1.0-2.6) in samples collected before starting the surgical day compared with 3 (range, 2-4; 95% CI, 2.0-3.3) at the end of the day (p = 0.004). In a separate study arm comprised of six loupes, 1 minute after being cleaned with an alcohol swab, bacterial presence on loupes decreased from a median of 2 (range, 2-3; 95% CI, 1.9-2.5) to a median of 1 (range, 0-2; 95% CI, 0.5-1.5; p = 0.012). Conclusions Loupes are a common reservoir for bacteria and fungi. Given the use of loupes directly over the surgical field and the lack of a barrier, care should be taken to decrease the bacterial load by cleaning loupes and airing out storage cases, which may decrease the risk of surgical field contamination and iatrogenic wound infections. Clinical Relevance Routine cleaning and disinfecting of optical loupes with alcohol pads can reduce microorganism colonization and should be implemented by surgeons who regularly use loupes in the operating room. Theoretically, particulate shedding from the loupes into the surgical field containing microorganisms could increase the risk of SSI, although this has not been proven clinically.
We have shown previously that creatine kinase (CK) activity is required for normal development an... more We have shown previously that creatine kinase (CK) activity is required for normal development and mineralization of chicken growth cartilage and that expression of the cytosolic isoforms of CK is related to the biosynthetic and energy status of the chondrocyte. In this study, we have characterized changes in isoenzyme activity and mRNA levels of CK (muscle-specific CK, M-CK; brain-type CK, B-CK; and mitochondrial CK subunits, MiaCK and MibCK) in the growth plate in situ and in chondrocyte culture systems that model the development/maturation program of the cartilage. The in vitro culture systems analyzed were as follows: tibial chondrocytes, which undergo hypertrophy; embryonic cephalic and caudal sternal chondrocytes, which differ from each other in their mineralization response to retinoic acid; and long-term micromass cultures of embryonic limb mesenchymal cells, which recapitulate the chondrocyte differentiation program. In all systems analyzed, B-CK was found to be the predominant isoform. In the growth plate, B-CK expression was highest in the most calcified regions, and M-CK was less abundant than B-CK in all regions of the growth plate. In tibial chondrocytes, an increase in B-CK expression was seen when the cells became hypertrophic. Expression of B-CK increased slightly over 15 days in mineralizing, retinoic acid-treated cephalic chondrocytes, but it decreased in nonmineralizing caudal chondrocytes, while there was little expression of M-CK. Interestingly, in limb mesenchyme cultures, significant M-CK expression was detected during chondrogenesis (days 2-7), whereas hypertrophic cells expressed only B-CK. Finally, expression of MiaCK and MibCK was low both in situ and in vitro. These observations suggest that the CK genes are differentially regulated during cartilage development and maturation and that an increase in CK expression is important in initiating chondrocyte maturation.
We have previously shown that thyroid stimulating hormone-β (TSHβ) mRNA levels are modulated by v... more We have previously shown that thyroid stimulating hormone-β (TSHβ) mRNA levels are modulated by vitamin A status in vivo and using transient transfection, that suppression of rat TSHβ gene promoter activity by all-trans retinoic acid (RA) requires RA receptor (RAR) and retinoid X receptor (RXR). In this paper we have used deletion analysis to delineate the sequences of the rTSHβ
Many surgical procedures require the placement of an inert or tissue-derived implant deep within ... more Many surgical procedures require the placement of an inert or tissue-derived implant deep within the body cavity. While the majority of these implants do not become colonized by bacteria, a small percentage develops a biofilm layer that harbors invasive microorganisms. In orthopaedic surgery, unresolved periprosthetic infections can lead to implant loosening, arthrodeses, amputations and sometimes death. The focus of this review is to describe development of an implant in which an antibiotic tethered to the metal surface is used to prevent bacterial colonization and biofilm formation. Building on well-established chemical syntheses, studies show that antibiotics can be linked to titanium through a self-assembled monolayer of siloxy amines. The stable metal-antibiotic construct resists bacterial colonization and biofilm formation while remaining amenable to osteoblastic cell adhesion and maturation. In an animal model, the antibiotic modified implant resists challenges by bacteria that are commonly present in periprosthetic infections. While the long-term efficacy and stability is still to be established, ongoing studies support the view that this novel type of bioactive surface has a real potential to mitigate or prevent the devastating consequences of orthopaedic infection.
Retinoic acid (RA) is important for normal mammalian development and growth. Ornithine decarboxyl... more Retinoic acid (RA) is important for normal mammalian development and growth. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the biosynthesis of the polyamines, and we have previously shown that ODC mRNA levels are suppressed by RA in human skin cells. Using HeLa cells, we now show that treatment with 0.5 fM RA for 24 h suppresses endogenous ODC mRNA levels and the expression of a transfected ODC/chloramphenicol acetyltransferase plasmid (Kpn-ODCCAT), containing sequences from-1450 to + 810 of the human ODC gene. Co-transfection with either the a-RA receptor (a-RAR) or a chimeric a-RA/oestrogen receptor (a-RAER) followed by treatment with the cognate hormone suppresses expression of Kpn-ODCCAT and Not-ODCCAT, which con
Journal of the American Academy of Orthopaedic Surgeons, Apr 1, 2015
ABSTRACT Orthopaedic infections are complex conditions that require immediate diagnosis and accur... more ABSTRACT Orthopaedic infections are complex conditions that require immediate diagnosis and accurate identification of the causative organisms to facilitate appropriate management. Conventional methodologies for diagnosis of these infections sometimes lack accuracy or sufficient rapidity. Molecular diagnostics is an emerging area of bench-to-bedside research in orthopaedic infections. Examples of promising molecular diagnostics include measurement of a specific biomarker in the synovial fluid, polymerase chain reaction-based detection of bacterial genes, and metabolomic determination of responses to orthopaedic infection. Copyright 2015 by the American Academy of Orthopaedic Surgeons.
Clinical Orthopaedics and Related Research, Aug 1, 2007
Results presented are not the statement or policy of the funding agencies. Each author certifies ... more Results presented are not the statement or policy of the funding agencies. Each author certifies that his or her institution has approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.
Clinical Orthopaedics and Related Research, May 15, 2019
We would like to correct errors found in our published study, “Surgical Loupes Worn by Orthopaedi... more We would like to correct errors found in our published study, “Surgical Loupes Worn by Orthopaedic Surgeons Are a Reservoir for Microorganisms”. In the Statistical Analysis section, we wrote: “Additionally, we used the Student’s t-test and the Wilcoxon signed-rank test to detect differences in categorical bacterial growth between paired samples.” Using a Student’s t-test or Wilcoxon tests is not adequate for the analysis given that these were not continuous but rather categorical variables. After a statistical consultation, we determined that the better statistical test in this setting is the Mann-Whitney U test. Given that the data does not change, the medians and 95% confidence intervals for the comparisons (before and after surgical day, before and after alcohol wipe) do not change. The statistical results of the Mann-Whitney U test are as follows: For the before and after surgical day comparison, analysis of the data sets with the Mann-Whitney U test yields a U-value of 20 and given that the critical value of U at p < 0.05 is 20, the result remains significant at p < 0.05. The z-score is 1.77604, yielding a p = 0.038. For the before and after alcohol wipe comparison, analysis of the data sets with the Mann-Whitney U test yields a U-value of 2.5 and given that the critical value of U at p < 0.05 is 5, the result remains significant at p < 0.05. The z-score is 2.40192, yielding a p = 0.02. As a result, our differences between these groups for both comparisons remain statistically different, although the p values change. Additionally, in the Discussion section, we wrote: “Lastly, we did not conduct clinical followup on the patients included in this study, and we describe a potential association of pathogen colonization with SSIs without demonstrating causation.” We intended to remove this sentence in the course of editing the paper but did not do so. The correct sentence should be: “Lastly, although particulate shedding from the loupes into the surgical field containing microorganisms could increase the risk of SSI, we did not assess SSI in the current study. Future studies should seek to evaluate the degree to which shedding of this sort might be clinically important, and if it is, to identify the best ways to mitigate it.” Finally, there was a typographical error in Table 1. Loupes of 2.5 magnification were used by 15 surgeons and those of 3.5 magnification were used by six surgeons, for a total of 21 surgeons. The authors apologize for the errors. Erratum to: Clin Orthop Relat Res DOI: 10.1097/CORR. 0000000000000651. The online version of this article can be found under DOI: 10.1097/CORR.0000000000000651.
Infection associated with inert implants is complicated by bacterial biofilm formation that rende... more Infection associated with inert implants is complicated by bacterial biofilm formation that renders the infection antibiotic insensitive. The goal of this investigation was to synthesize and characterize a vancomycin (VAN)modified bone allograft that could render the tissue inhospitable to bacterial colonization and the establishment of infection. We found that the numbers of primary amines, which could serve as anchors for chemical synthesis, increased with limited demineralization. Using these amines, we coupled two linkers and VAN to bone using Fmoc chemistry. By immunohistochemistry, VAN was abundant on the surface of the allograft; based on elution and measurement of bound antibody, this coupling yielded at least *26 ng VAN=mg bone. The coupled VAN appeared to be permanently bound to the allograft, as it showed no elution in a disk diffusion assay, and, importantly, resisted colonization by Staphylococcus aureus challenges. We suggest that this chimeric construct represents a new generation of antibiotic-modified allografts that provide antibacterial properties.
Antimicrobial Agents and Chemotherapy, Feb 1, 2011
Infection is an important medical problem associated with the use of bone allografts. To retard b... more Infection is an important medical problem associated with the use of bone allografts. To retard bacterial colonization, we have recently reported on the modification of bone allografts with the antibiotic vancomycin (VAN). In this report, we examine the ability of this antibiotic-modified allograft to resist bacterial colonization and biofilm formation. When antibiotic was coupled to the allograft, a uniform distribution of the antibiotic was apparent. Following challenges with Staphylococcus aureus for 6 h, the covalently bonded VAN decreased colonization as a function of inoculum, ranging from 0.8 to 2.0 log 10 CFU. Furthermore, the VAN-modified surface resisted biofilm formation, even in topographical niches that provide a protected environment for bacterial adhesion. Attachment of the antibiotic to the allograft surface was robust, and the bonded VAN was stable whether incubated in aqueous media or in air, maintaining levels of 75 to 100% of initial levels over 60 days. While the VAN-modified allograft inhibited the Gram-positive S. aureus colonization, in keeping with VANs spectrum of activity, the VAN-modified allograft was readily colonized by the Gram-negative Escherichia coli. Finally, initial toxicity measures indicated that the VAN-modified allograft did not influence osteoblast colonization or viability. Since the covalently tethered antibiotic is stable, is active, retains its specificity, and does not exhibit toxicity, it is concluded that this modified allograft holds great promise for decreasing bone graft-associated infections.
Introduction: The purpose of this study was to evaluate bacterial adherence to common casting mat... more Introduction: The purpose of this study was to evaluate bacterial adherence to common casting materials including plaster of Paris (plaster), fiberglass, three-dimensional (3D) printed plastic, and siliconecoated 3D printed plastic. Methods: The minimal inhibitory concentration of a phosphate-free detergent (Palmolive) needed to achieve total bacterial kill off was determined. 3D printed polylactic acid plastic samples were coated with silicone. Plaster, fiberglass, plastic, and silicone-coated plastic samples were inoculated with Staphylococcus aureus. After bacterial inoculation, scanning electron microscopy of the samples was performed to visualize bacterial adherence to the materials' surface. Using either sterile water or a 5% detergent solution, the materials were subjected to washings. Each material was run in 30 replicates: 6 without washing, 6 with sterile water for 1 minute, 6 with detergent for 1 minute, 6 with sterile water for 3 minutes, and 6 with detergent for 3 minutes. The replicates that did not undergo a washing trial represented the initial bacterial inoculation. Samples were then rinsed and sonicated in polysorbate to isolate the remaining adherent bacteria on the materials' surface. The sonicated solutions were plated, incubated, and counted for quantification of colony forming units (CFU) of bacteria. This protocol was repeated for a total of four trials. Results: During inoculation, there were significantly less bacteria that adhered to silicone-coated 3D printed plastic (58879 CFU) compared to plastic (217479 CFU), plaster (140063 CFU), and fiberglass (550546 CFU). Silicone coating showed further superiority. Silicone-coated 3D printed plastic was able to be decontaminated as demonstrated by significantly fewer remaining bacteria (9.3%) on its surface after being washed with a 5% detergent solution (1797 CFU) compared to sterile water (19321 CFU). The mean remaining bacteria on silicone-coated 3D printed plastic was significantly less than that remaining on all other materials when washed with either sterile water or a detergent solution for both durations of 1 minute and 3 minutes. Conclusions: The current study demonstrates that significantly less bacteria adhere to the surface of 3D printed plastic with silicone coating showing added protection and that this material can be decontaminated to a greater degree with washing than conventional casting materials. These results provide evidence that 3D printed casts can be washed and successfully decontaminated during a patient's period of immobilization, which is advantageous especially during an infectious crisis such as the coronavirus disease 2019 (COVID-19) pandemic.
Changes in cell shape occur during the cell cycle and influence cell proliferation and differenti... more Changes in cell shape occur during the cell cycle and influence cell proliferation and differentiation. In order to study how altered cell proliferation and cell shape are interrelated, we have studied ornithine decarboxylase (ODC) regulation in cultured normal human epidermal keratinocytes (NHEK). Cytoskeletal disruptors have been reported to modulate regulation of ODC; the products of ODC, the polyamines, influence actin polymerization rates in vitro, and polyamine auxotrophs have profoundly disrupted cytoskeletons. Therefore, altered ODC levels could be involved in signaling changes in cell shape and an intact cytoskeleton could transduce signals to regulate ODC levels. We had previously observed that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which profoundly alters cell shape, markedly suppresses ODC biosynthesis in NHEK solely at posttranscriptional/protein synthesis levels. TPA treatment caused NHEK to rapidly assume a rounded morphology that was accompanied by a change in actin organization, as determined by rhodamine-phalloidin labeling. Immunolocalization of ODC showed a perinuclear/nuclear distribution in untreated NHEK and a more diffuse pattern after TPA treatment that was apparent within 15-30 min. Changes in ODC enzyme activity are not significant until 60 min after TPA treatment. NHEK treated with cytochalasin B or D to inhibit actin polymerization exhibited a diffuse ODC localization that could be reversed by removal of the cytochalasin; inhibition of ODC by alpha-difluoromethylornithine caused a diffuse ODC localization. All treatments resulted in cytoskeletal remodeling. These data are the first evidence for a distinct subcellular localization for ODC and suggest that changes in ODC localization may be an initial step in regulation of ODC activity. Furthermore, changes in ODC activity cause an altered cytoskeleton, suggesting one means by which growth regulatory signals can be transduced to the cytoskeleton from various signaling pathways.
Bioorganic & Medicinal Chemistry Letters, May 1, 2007
Self-protecting Ti6Al4V alloy pins were prepared by covalent bonding of bis(ethylene glycol) link... more Self-protecting Ti6Al4V alloy pins were prepared by covalent bonding of bis(ethylene glycol) linkers, then vancomycin to the oxidized, aminopropylated Ti6Al4V alloy surface. Fluorescence modification-enabled estimation of yields of free amines on the metallic surface monolayer at each reaction step. The vancomycin-protected Ti6Al4V pins were not colonized by Staphylococcus aureus, even after 44 days storage in physiological buffer. These results provide a basis for testing self-protection against S. aureus colonization in animal models.
Clinical Orthopaedics and Related Research, Feb 6, 2019
Background Surgeons frequently use optical loupes to magnify the surgical field; they are typical... more Background Surgeons frequently use optical loupes to magnify the surgical field; they are typically unprotected when positioned directly over the wound, where particulate shedding containing microorganisms could potentially lead to surgical site infections (SSIs). SSIs are rare in some orthopaedic subspecialties such as hand surgery; however, in other subspecialties, for example, the spine, where surgeons often use loupes, SSIs can have devastating consequences. Questions/purposes (1) What is the degree of bacterial and fungi organism colonization of surgical loupes and storage cases? (2) Is there a difference in the degree of colonization at the beginning and the end of a surgery day? (3) Does an alcohol swab reduce bacterial colonization of surgical loupes? Methods The surgical loupes of 21 orthopaedic surgeons from a large, regional orthopaedic practice were cultured over a 3-month period and form the basis of this study. Five loupe storage cases were also cultured. In two different subgroup comparisons, the presence of microorganisms was evaluated just before the start and immediately after the end of the surgical day (n = 9) and before and 1 minute after cleaning with an alcohol swab (n = 6). A total of 36 cultures were evaluated. Surgeons who declined to participate in the study were excluded. The number of loupes selected for all of the analyses were samples of convenience and limited by surgeon availability. The degree of bacterial and fungal presence was graded using a point system: 0 = no growth; 1 = limited growth (meaning few scattered colonies); 2 = moderate growth; 3 = extensive but scattered growth; and 4 = growth consuming the entire plate. Demographic data were assessed using descriptive statistics. Additionally, the Student’s t and Wilcoxon signed-rank tests were used to detect differences in categorical bacterial growth between paired samples. A p value of 0.05 represented statistical significance. Kappa statistics of reliability were performed to evaluate interobserver agreement of microorganism growth in the culture plates. Results Bacteria were present in 19 of 21 (90%) sets of loupes. Five species of bacteria were noted. Fungi were present in 10 of 21 (48%) sets of loupes. Bacterial contamination was identified in two storage cases (40%) and fungi were present in five cases (100%). In a subset of nine loupes tested, the degree of bacterial presence had a median of 2 (range, 1-4; 95% confidence interval [CI], 1.0-2.6) in samples collected before starting the surgical day compared with 3 (range, 2-4; 95% CI, 2.0-3.3) at the end of the day (p = 0.004). In a separate study arm comprised of six loupes, 1 minute after being cleaned with an alcohol swab, bacterial presence on loupes decreased from a median of 2 (range, 2-3; 95% CI, 1.9-2.5) to a median of 1 (range, 0-2; 95% CI, 0.5-1.5; p = 0.012). Conclusions Loupes are a common reservoir for bacteria and fungi. Given the use of loupes directly over the surgical field and the lack of a barrier, care should be taken to decrease the bacterial load by cleaning loupes and airing out storage cases, which may decrease the risk of surgical field contamination and iatrogenic wound infections. Clinical Relevance Routine cleaning and disinfecting of optical loupes with alcohol pads can reduce microorganism colonization and should be implemented by surgeons who regularly use loupes in the operating room. Theoretically, particulate shedding from the loupes into the surgical field containing microorganisms could increase the risk of SSI, although this has not been proven clinically.
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Papers by Noreen Hickok