RESUMO
BACKGROUND: Crude and adjusted mortality rates for patients with non-ventilator hospital-acquired pneumonia (NV-HAP) are amongst the highest of all healthcare-associated infections, leading to calls for greater prevention. Patients prone to NV-HAP, however, tend to be severely ill at baseline making it unclear whether their high mortality rates are due to NV-HAP, underlying conditions, or both. METHODS: Two infectious disease physicians conducted detailed medical record reviews on 150 randomly selected adults from 4 hospitals who died in-hospital following an NV-HAP event between April 2016 and May 2021. Reviewers abstracted risk factors, estimated the preventability of NV-HAP, identified causes of death, and adjudicated the preventability of death. RESULTS: Patients' median age was 69.3 (IQR 60.7-77.4) and 43.3% were female. Comorbidities were common: 57% had cancer, 30% chronic kidney disease, 29% chronic lung disease, and 27% heart failure. At least one hospice-eligible condition was present before NV-HAP in 54% and "Do Not Resuscitate" orders in 24%. Most (99%) had difficult-to-modify NV-HAP risk factors: 76% altered mental status, 35% dysphagia, and 27% nasogastric/orogastric tubes. NV-HAP was deemed possibly or probably preventable in 21% and hospital death likely or very likely preventable in 8.6%. CONCLUSIONS: Most patients who die following NV-HAP have multiple, severe underlying comorbidities and difficult-to-modify risk factors for NV-HAP. Only 1 in 5 NV-HAPs that culminated in death and 1 in 12 deaths following NV-HAP were judged potentially preventable. This does not diminish the importance of NV-HAP prevention programs but informs expectations about the potential magnitude of their impact on hospital deaths.
RESUMO
Staphylococcus aureus bacteremia (SAB) remains a clinically challenging infection despite extensive investigation. Repurposing medications approved for other indications is appealing as clinical safety profiles have already been established. Ticagrelor, a reversible adenosine diphosphate receptor antagonist that prevents platelet aggregation, is indicated for patients suffering from acute coronary syndrome (ACS). However, some clinical data suggest that patients treated with ticagrelor are less likely to have poor outcomes due to S. aureus infection. There are several potential mechanisms by which ticagrelor may affect S. aureus virulence. These include direct antibacterial activity, up-regulation of the innate immune system through boosting platelet-mediated S. aureus killing, and prevention of S. aureus adhesion to host tissues. In this Pearl, we review the clinical data surrounding ticagrelor and infection as well as explore the evidence surrounding these proposed mechanisms of action. While more evidence is needed before antiplatelet medications formally become part of the arsenal against S. aureus infection, these potential mechanisms represent exciting pathways to target in the host/pathogen interface.
Assuntos
Bacteriemia/tratamento farmacológico , Plaquetas/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Ticagrelor/uso terapêutico , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Inibidores da Agregação Plaquetária/uso terapêutico , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologiaRESUMO
BACKGROUND: The risk of periprosthetic joint infection (PJI) is higher in persons who inject drugs (PWID) after total joint arthroplasty (TJA), though reported rates vary widely. This study was designed to assess outcomes of TJA in PWID and to describe factors associated with improved PJI outcomes among PWID. METHODS: A retrospective matched cohort study was performed using a 1:4 match among those with and those without a history of injection drug use (IDU) undergoing TJA. Demographic, surgical, and outcome variables were compared in multivariate logistic regressions to determine PJI predictors. Kaplan-Meier analyses were constructed to characterize the difference in survival of patients who did not have PJI or undergo joint explantation between PWID and the matching cohort. RESULTS: PWID had a 9-fold increased risk of PJI compared to the matched cohort (odds ratio 9.605, 95% CI 2.781-33.175, P < .001). Ten of 17 PWID whose last use was within 6 months (active use) of primary TJA had a PJI, while 7 of 41 PWID who did not have active use developed a PJI. Of PWID with PJI, treatment failure was seen in 15 of 17, while in patients who did not have an IDU history, 5 of 8 with PJI had treatment failure. CONCLUSION: IDU is a significant risk factor for PJI following TJA. Future work investigating the effect of a multidisciplinary support team to assist in cessation of IDU and to provide social support may improve outcomes and reduce morbidity in this vulnerable population.
Assuntos
Artrite Infecciosa , Artroplastia de Quadril , Artroplastia do Joelho , Usuários de Drogas , Infecções Relacionadas à Prótese , Abuso de Substâncias por Via Intravenosa , Humanos , Infecções Relacionadas à Prótese/etiologia , Infecções Relacionadas à Prótese/complicações , Artroplastia do Joelho/efeitos adversos , Artroplastia de Quadril/efeitos adversos , Estudos Retrospectivos , Estudos de Coortes , Abuso de Substâncias por Via Intravenosa/complicações , Artrite Infecciosa/etiologia , Fatores de RiscoRESUMO
Large mandibular defects are clinically challenging to reconstruct due to the complex anatomy of the jaw and the limited availability of appropriate tissue for repair. We envision leveraging current advances in fabrication and biomaterials to create implantable devices that generate bone within the patients themselves suitable for their own specific anatomical pathology. The in vivo bioreactor strategy facilitates the generation of large autologous vascularized bony tissue of customized geometry without the addition of exogenous growth factors or cells. To translate this technology, we investigated its success in reconstructing a mandibular defect of physiologically relevant size in sheep. We fabricated and implanted 3D-printed in vivo bioreactors against rib periosteum and utilized biomaterial-based space maintenance to preserve the native anatomical mandibular structure in the defect site before reconstruction. Nine weeks after bioreactor implantation, the ovine mandibles were repaired with the autologous bony tissue generated from the in vivo bioreactors. We evaluated tissues generated in bioreactors by radiographic, histological, mechanical, and biomolecular assays and repaired mandibles by radiographic and histological assays. Biomaterial-aided mandibular reconstruction was successful in a large superior marginal defect in five of six (83%) sheep. Given that these studies utilized clinically available biomaterials, such as bone cement and ceramic particles, this strategy is designed for rapid human translation to improve outcomes in patients with large mandibular defects.
Assuntos
Substitutos Ósseos , Mandíbula , Traumatismos Mandibulares , Periósteo , Impressão Tridimensional , Engenharia Tecidual , Animais , Reatores Biológicos , Feminino , Mandíbula/metabolismo , Mandíbula/patologia , Traumatismos Mandibulares/metabolismo , Traumatismos Mandibulares/patologia , Traumatismos Mandibulares/terapia , Periósteo/metabolismo , Periósteo/patologia , OvinosRESUMO
Total joint arthroplasty is an important therapeutic option for patients suffering from osteoarthritis and other degenerative joint diseases. However, joint replacements are susceptible to periprosthetic joint infection especially by staphylococci and other gram-positive organisms. Antibiotic prophylaxis, or systemic administration of antibiotics prior to primary arthroplasty, has been shown to reduce rates of surgical site infection and periprosthetic joint infection. The motivation and goals behind antibiotic prophylaxis, current guidelines, the choice of antibiotic agents, and important factors in antimicrobial administration, including its dose, timing, and duration, are reviewed.
Assuntos
Artroplastia de Quadril , Artroplastia de Substituição , Infecções Relacionadas à Prótese , Antibacterianos/uso terapêutico , Antibioticoprofilaxia , Artroplastia de Substituição/efeitos adversos , Artroplastia de Quadril/efeitos adversos , Humanos , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Relacionadas à Prótese/prevenção & controle , Infecção da Ferida Cirúrgica/tratamento farmacológico , Infecção da Ferida Cirúrgica/prevenção & controleRESUMO
Mucormycosis, also known as zygomycosis, is an aggressive infection caused by a ubiquitous group of molds known as mucormycetes and is often associated with immune suppression or trauma among immunocompetent populations. We present the case of a 19-year-old woman who was involved in a motor vehicle accident in whom rapidly progressive invasive cutaneous facial mucormycosis subsequently developed. The diagnosis, treatment options, and incidence of this disease process are discussed in the context of trauma.
Assuntos
Traumatismos Faciais/microbiologia , Mucormicose/diagnóstico , Acidentes de Trânsito , Anfotericina B/uso terapêutico , Antifúngicos/uso terapêutico , Biomarcadores/análise , Terapia Combinada , Diagnóstico Diferencial , Traumatismos Faciais/tratamento farmacológico , Traumatismos Faciais/cirurgia , Evolução Fatal , Feminino , Humanos , Mucormicose/tratamento farmacológico , Mucormicose/cirurgia , Adulto JovemRESUMO
While antibiotic-eluting polymethylmethacrylate space maintainers have shown efficacy in the treatment of bacterial periprosthetic joint infection and osteomyelitis, antifungal-eluting space maintainers are associated with greater limitations for treatment of fungal musculoskeletal infections including limited elution concentration and duration. In this study, we have designed a porous econazole-eluting space maintainer capable of greater inhibition of fungal growth than traditional solid space maintainers. The eluted econazole demonstrated bioactivity in a concentration-dependent manner against the most common species responsible for fungal periprosthetic joint infection as well as staphylococci. Lastly, these porous space maintainers retain compressive mechanical properties appropriate to maintain space before definitive repair of the joint or bony defect.
Assuntos
Antifúngicos/química , Materiais Biocompatíveis , Econazol/química , Micoses/tratamento farmacológico , Infecções Relacionadas à Prótese/tratamento farmacológico , Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Econazol/farmacologia , Teste de Materiais , Polimetil Metacrilato , Porosidade , Staphylococcus aureus/efeitos dos fármacosRESUMO
BACKGROUND: Azole-resistant aspergillosis in high-risk patients with hematological malignancy or hematopoietic stem cell transplantation (HSCT) is a cause of concern. METHODS: We examined changes over time in triazole minimum inhibitory concentrations (MICs) of 290 sequential Aspergillus isolates recovered from respiratory sources during 1999-2002 (before introduction of the Aspergillus-potent triazoles voriconazole and posaconazole) and 2003-2015 at MD Anderson Cancer Center. We also tested for polymorphisms in ergosterol biosynthetic genes (cyp51A, erg3C, erg1) in the 37 Aspergillus fumigatus isolates isolated from both periods that had non-wild-type (WT) MICs. For the 107 patients with hematologic cancer and/or HSCT with invasive pulmonary aspergillosis, we correlated in vitro susceptibility with 42-day mortality. RESULTS: Non-WT MICs were found in 37 (13%) isolates and was only low level (MIC <8 mg/L) in all isolates. Higher-triazole MICs were more frequent in the second period and were Aspergillus-species specific, and only encountered in A. fumigatus. No polymorphisms in cyp51A, erg3C, erg1 genes were identified. There was no correlation between in vitro MICs with 42-day mortality in patients with invasive pulmonary aspergillosis, irrespective of antifungal treatment. Asian race (odds ratio [OR], 20.9; 95% confidence interval [CI], 2.5-173.5; P = .005) and azole exposure in the prior 3 months (OR, 9.6; 95% CI, 1.9-48.5; P = .006) were associated with azole resistance. CONCLUSIONS: Non-WT azole MICs in Aspergillus are increasing and this is associated with prior azole exposure in patients with hematologic cancer or HSCT. However, no correlation of MIC with outcome of aspergillosis was found in our patient cohort.
Assuntos
Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Aspergilose Pulmonar Invasiva/microbiologia , Atenção Terciária à Saúde , Triazóis/farmacologia , Adulto , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergillus/genética , Aspergillus/isolamento & purificação , Aspergillus fumigatus/efeitos dos fármacos , Estudos de Coortes , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica/genética , Ergosterol/biossíntese , Feminino , Proteínas Fúngicas/genética , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/microbiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Aspergilose Pulmonar Invasiva/tratamento farmacológico , Aspergilose Pulmonar Invasiva/mortalidade , Masculino , Testes de Sensibilidade Microbiana , Polimorfismo Genético , Estudos Prospectivos , Resultado do Tratamento , Triazóis/uso terapêutico , Voriconazol/farmacologia , Voriconazol/uso terapêutico , Adulto JovemRESUMO
In this work, we describe the synthesis and characterization of variants of poly(diol fumarate) and poly(diol fumarate-co-succinate). Through a Fischer esterification, α,ω-diols and dicarboxylic acids were polymerized to form aliphatic polyester comacromers. Because of the carbon-carbon double bond of fumaric acid, incorporating it into the macromer backbone structure resulted in unsaturated chains. By choosing α,ω-diols of different lengths (1,6-hexanediol, 1,8-octanediol, and 1,10-decanediol) and controlling the amount of fumaric acid in the dicarboxylic acid monomer feed (33, 50, and 100 mol %), nine diol-based macromer variants were synthesized and characterized for molecular weight, number of unsaturated bonds per chain, and thermal properties. Degradation and in vitro cytotoxicity were also measured in a subset of macromers. As proof-of-principle, macromer networks were photo-cross-linked to demonstrate the ability to perform free radical addition using the unsaturated macromer backbone. Cross-linked macromer networks were also characterized for physicochemical properties (swelling, sol fraction, compressive modulus) based on diol length and amount of unsaturated bonds. A statistical model was built using data generated from these diol-based macromers and macromer networks to evaluate the impact of monomer inputs on final macromer and macromer network properties. With the ability to be modified by free radical addition, biodegradable unsaturated polyesters serve as important macromers in the design of devices such as drug delivery vehicles and tissue scaffolds. Given the ability to extensively control final macromer properties based on monomer input parameters, poly(diol fumarate) and poly(diol fumarate-co-succinate) represent an exciting new class of macromers.
Assuntos
Fumaratos/síntese química , Glicóis/síntese química , Poliésteres/síntese química , Succinatos/síntese química , Força Compressiva , Sistemas de Liberação de Medicamentos , Fumaratos/química , Glicóis/química , Humanos , Luz , Peso Molecular , Poliésteres/química , Succinatos/química , Alicerces Teciduais , MolhabilidadeRESUMO
BACKGROUND: Mucormycosis is a destructive invasive mold infection afflicting patients with diabetes and hematologic malignancies. Patients with diabetes are often treated with statins, which have been shown to have antifungal properties. We sought to examine the effects of statins on Rhizopus oryzae, a common cause of mucormycosis. METHODS: Clinical strains of R. oryzae were exposed to lovastatin, atorvastatin, and simvastatin and the minimum inhibitory concentrations (MICs) were determined. R. oryzae germination, DNA fragmentation, susceptibility to oxidative stress, and ability to damage endothelial cells were assessed. We further investigated the impact of exposure to lovastatin on the virulence of R. oryzae RESULTS: All statins had MICs of >64 µg/mL against R. oryzae Exposure of R. oryzae to statins decreased germling formation, induced DNA fragmentation, and attenuated damage to endothelial cells independently of the expression of GRP78 and CotH. Additionally, R. oryzae exposed to lovastatin showed macroscopic loss of melanin, yielded increased susceptibility to the oxidative agent peroxide, and had attenuated virulence in both fly and mouse models of mucormycosis. CONCLUSIONS: Exposure of R. oryzae to statins at concentrations below their MICs decreased virulence both in vitro and in vivo. Further investigation is warranted into the use of statins as adjunctive therapy in mucormycosis.
Assuntos
Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Mucormicose/tratamento farmacológico , Rhizopus/efeitos dos fármacos , Virulência/efeitos dos fármacos , Animais , Atorvastatina/farmacologia , Atorvastatina/uso terapêutico , Dípteros/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Lovastatina/farmacologia , Lovastatina/uso terapêutico , Camundongos , Testes de Sensibilidade Microbiana , Sinvastatina/farmacologia , Sinvastatina/uso terapêutico , Esporos Fúngicos/efeitos dos fármacos , TexasRESUMO
Infection is one of the most common complications associated with medical interventions and implants. As tissue engineering strategies to replace missing or damaged tissue advance, the focus on prevention and treatment of concomitant infection has also begun to emerge as an important area of research. Because the in vivo environment is a complex interaction between host tissue, implanted materials, and native immune system that cannot be replicated in vitro, animal models of infection are integral in evaluating the safety and efficacy of experimental treatments for infection. In this review, considerations for selecting an animal model, established models of infection, and areas that require further model development are discussed with regard to cutaneous, fascial, and orthopedic infections.
Assuntos
Infecções/terapia , Engenharia Tecidual/métodos , Animais , Doenças do Tecido Conjuntivo/terapia , Modelos Animais de Doenças , Herniorrafia/métodos , Especificidade de Hospedeiro , Humanos , Osteomielite/terapia , Dermatopatias Infecciosas/terapiaRESUMO
Craniofacial defects require a treatment approach that provides both robust tissues to withstand the forces of mastication and high geometric fidelity that allows restoration of facial architecture. When the surrounding soft tissue is compromised either through lack of quantity (insufficient soft tissue to enclose a graft) or quality (insufficient vascularity or inducible cells), a vascularized construct is needed for reconstruction. Tissue engineering using customized 3D printed bioreactors enables the generation of mechanically robust, vascularized bony tissues of the desired geometry. While this approach has been shown to be effective when utilized for reconstruction of non-load bearing ovine angular defects and partial segmental defects, the two-stage approach to mandibular reconstruction requires testing in a large, load-bearing defect. In this study, 5 sheep underwent bioreactor implantation and the creation of a load-bearing mandibular defect. Two bioreactor geometries were tested: a larger complex bioreactor with a central groove, and a smaller rectangular bioreactor that were filled with a mix of xenograft and autograft (initial bone volume/total volume BV/TV of 31.8 ± 1.6%). At transfer, the tissues generated within large and small bioreactors were composed of a mix of lamellar and woven bone and had BV/TV of 55.3 ± 2.6% and 59.2 ± 6.3%, respectively. After transfer of the large bioreactors to the mandibular defect, the bioreactor tissues continued to remodel, reaching a final BV/TV of 64.5 ± 6.2%. Despite recalcitrant infections, viable osteoblasts were seen within the transferred tissues to the mandibular site at the end of the study, suggesting that a vascularized customized bony flap is a potentially effective reconstructive strategy when combined with an optimal stabilization strategy and local antibiotic delivery prior to development of a deep-seated infection.
Assuntos
Osteotomia Mandibular , Procedimentos de Cirurgia Plástica , Humanos , Animais , Ovinos , Engenharia Tecidual , Retalhos Cirúrgicos/cirurgia , Mandíbula/cirurgia , Transplante ÓsseoRESUMO
Epithelia provide the first line of defense against foreign pathogens, and disruption of tissue homeostasis frequently allows for opportunistic infections. Here we provide a protocol for induction of epithelial cell loss in zebrafish larvae, followed by infection with fungal pathogens. Details are provided for monitoring larval survival after infection, assessment of fungal burden, and prophylactic treatment with antifungal compounds. Limitations of the protocol include potential antifungal toxicity and high fungal inoculums to induce lethal infection with some pathogenic fungal species. For complete details on the use and execution of this protocol, please refer to Wurster et al. (2021).
Assuntos
Modelos Animais de Doenças , Células Epiteliais/patologia , Larva/microbiologia , Micoses , Peixe-Zebra/microbiologia , Animais , Antifúngicos/uso terapêutico , Micoses/tratamento farmacológico , Micoses/microbiologia , Micoses/patologiaRESUMO
Severe and often fatal opportunistic fungal infections arise frequently following mucosal damage caused by trauma or cytotoxic chemotherapy. Interaction of fungal pathogens with epithelial cells that comprise mucosae is a key early event associated with invasion, and, therefore, enhancing epithelial defense mechanisms may mitigate infection. Here, we establish a model of mold and yeast infection mediated by inducible epithelial cell loss in larval zebrafish. Epithelial cell loss by extrusion promotes exposure of laminin associated with increased fungal attachment, invasion, and larval lethality, whereas fungi defective in adherence or filamentation have reduced virulence. Transcriptional profiling identifies significant upregulation of the epidermal growth factor receptor ligand epigen (EPGN) upon mucosal damage. Treatment with recombinant human EPGN suppresses epithelial cell extrusion, leading to reduced fungal invasion and significantly enhanced survival. These data support the concept of augmenting epithelial restorative capacity to attenuate pathogenic invasion of fungi associated with human disease.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Mucosa/microbiologia , Mucosa/patologia , Rhizopus/patogenicidade , Animais , Epigen/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Humanos , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Larva/microbiologia , Modelos Biológicos , Mucosa/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Proteínas Recombinantes/farmacologia , Rhizopus/ultraestrutura , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/fisiologia , Fatores de Tempo , Peixe-Zebra/microbiologiaRESUMO
In light of the current novel coronavirus (COVID-19) pandemic, as well as other viral outbreaks in the 21st century, there is a dire need for new diagnostic and therapeutic strategies to combat infectious diseases worldwide. As a convergence science, tissue engineering has traditionally focused on the application of engineering principles to biological systems, collaboration across disciplines, and rapid translation of technologies from the benchtop to the bedside. Given these strengths, tissue engineers are particularly well suited to apply their skill set to the current crisis and viral outbreaks in general. This work introduces the basics of virology and epidemiology for tissue engineers, and highlights important developments in the field of tissue engineering relevant to the current pandemic, including in vitro model systems, vaccine technology, and small-molecule drug delivery. COVID-19 serves as a call to arms for scientists across all disciplines, and tissue engineers are well trained to be leaders and contributors in this time of need. Impact statement Given the steep mortality caused by the recent novel coronavirus (COVID-19) pandemic, there is clear need for advances in diagnostics and therapeutics for viral outbreaks. Tissue engineering has the potential for critical impact on clinical outcomes in viral outbreaks. Tissue engineers, if mobilized, could play key roles as leaders in the outbreak, given their ability to apply engineering principles to biological processes, experience in collaborative environments, and penchant for technological translation from benchtop to bedside. In this work, three areas pioneered by tissue engineers that could be applied to the current COVID-19 crisis and future viral outbreaks are highlighted.
Assuntos
Betacoronavirus , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Engenharia Tecidual , COVID-19 , Teste para COVID-19 , Vacinas contra COVID-19 , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Sistemas de Liberação de Medicamentos , Humanos , Técnicas In Vitro , Pandemias/prevenção & controle , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/terapia , SARS-CoV-2 , Vacinas Virais , Tratamento Farmacológico da COVID-19RESUMO
The generation of vascularized mineralized tissues of complex geometry without the use of extrinsic growth factors or exogenous cells requires a large animal model to recapitulate the challenges seen in the clinic. The proposed versatile ovine model can be utilized to investigate the use of a customized bioreactor to generate mineralized tissue, matching the size and shape of a defect before transfer to and integration within another site. The protocol results in bioreactors that can be harvested for investigation of the effects of different biomaterials for the generation of bone or to generate tissues appropriate for repair of bony defects; this protocol focuses on reconstruction of the mandible but could be modified for orthopedic applications. The bioreactor packing material can be altered, allowing for the study of various commercially available or novel graft materials. The surgical procedure requires â¼1.5 h to implant four bioreactors adjacent to rib periosteum. After 9 weeks, the harvest of the bioreactor tissue takes approximately 1 h. If creating a craniofacial defect, an additional 2 h should be taken for mandibular defect creation and 2 to 3 h for the reconstruction. Sheep that have undergone reconstruction are typically euthanized after 12 weeks to allow for evaluation of transferred tissues. In this protocol, we discuss the necessary steps to ensure the reproducibility and analytical techniques to assess bone regeneration such as microcomputed tomography, mechanical analysis, and histology. Impact statement Bone grafting is a frequent procedure in the fields of orthopedics, otolaryngology, and oral and maxillofacial surgery. Generating customized, vascularized, and mechanically robust bony tissues while eliminating common complications such as donor site morbidity with autograft harvest or lack of suitable mechanical properties with commercially available synthetic graft would greatly improve the lives of patients. A large animal model is necessary to generate tissues of clinically relevant geometries. In this article, a reproducible ovine model of in vivo bioreactor technology toward customized bone generation is presented with broad application to tissue engineering and regenerative medicine.
Assuntos
Materiais Biocompatíveis/química , Reatores Biológicos , Doenças Ósseas/terapia , Regeneração Óssea , Transplante Ósseo/métodos , Modelos Animais , Engenharia Tecidual/métodos , Animais , OvinosRESUMO
Trauma-related necrotizing myocutaneous mucormycosis (NMM) has a high morbidity and mortality in victims of combat-related injuries, geometeorological disasters, and severe burns. Inspired by the observation that several recent clusters of NMM have been associated with extreme mechanical forces (e.g., during tornados), we studied the impact of mechanical stress on Mucoralean biology and virulence in a Drosophila melanogaster infection model. In contrast to other experimental procedures to exert mechanical stress, tornadic shear challenge (TSC) by magnetic stirring induced a hypervirulent phenotype in several clinically relevant Mucorales species but not in Aspergillus or Fusarium Whereas fungal growth rates, morphogenesis, and susceptibility to noxious environments or phagocytes were not altered by TSC, soluble factors released in the supernatant of shear-challenged R. arrhizus spores rendered static spores hypervirulent. Consistent with a rapid decay of TSC-induced hypervirulence, minimal transcriptional changes were revealed by comparative RNA sequencing analysis of static and shear-challenged Rhizopus arrhizus However, inhibition of the calcineurin/heat shock protein 90 (hsp90) stress response circuitry by cyclosporine and tanespimycin abrogated the increased pathogenicity of R. arrhizus spores following TSC. Similarly, calcineurin loss-of-function mutants of Mucor circinelloides displayed no increased virulence capacity in flies after undergoing TSC. Collectively, these results establish that TSC induces hypervirulence specifically in Mucorales and point out the calcineurin/hsp90 pathway as a key orchestrator of this phenotype. Our findings invite future studies of topical calcineurin inhibitor treatment of wounds as an adjunct mitigation strategy for NMM following high-energy trauma.IMPORTANCE Given the limited efficacy of current medical treatments in trauma-related necrotizing mucormycosis, there is a dire need to better understand the Mucoralean pathophysiology in order to develop novel strategies to counteract fungal tissue invasion following severe trauma. Here, we describe that tornadic shear stress challenge transiently induces a hypervirulent phenotype in various pathogenic Mucorales species but not in other molds known to cause wound infections. Pharmacological and genetic inhibition of calcineurin signaling abrogated hypervirulence in shear stress-challenged Mucorales, encouraging further evaluation of (topical) calcineurin inhibitors to improve therapeutic outcomes of NMM after combat-related blast injuries or violent storms.
Assuntos
Calcineurina/metabolismo , Mucorales/patogenicidade , Estresse Mecânico , Células A549 , Animais , Drosophila melanogaster , Feminino , Fusarium/genética , Fusarium/patogenicidade , Humanos , Mucorales/genética , Mucormicose/tratamento farmacológico , Mucormicose/microbiologia , Fenótipo , Rhizopus oryzae/genética , Rhizopus oryzae/patogenicidade , Esporos Fúngicos/genética , Esporos Fúngicos/patogenicidade , VirulênciaRESUMO
Mandibular reconstruction requires functional and aesthetic repair and is further complicated by contamination from oral and skin flora. Antibiotic-releasing porous space maintainers have been developed for the local release of vancomycin and to promote soft tissue attachment. In this study, mandibular defects in six sheep were inoculated with 106 colony forming units of Staphylococcus aureus; three sheep were implanted with unloaded porous space maintainers and three sheep were implanted with vancomycin-loaded space maintainers within the defect site. During the same surgery, 3D-printed in vivo bioreactors containing autograft or xenograft were implanted adjacent to rib periosteum. After 9 weeks, animals were euthanized, and tissues were analyzed. Antibiotic-loaded space maintainers were able to prevent dehiscence of soft tissue overlying the space maintainer, reduce local inflammatory cells, eliminate the persistence of pathogens, and prevent the increase in mandibular size compared to unloaded space maintainers in this sheep model. Animals with an untreated mandibular infection formed bony tissues with greater density and maturity within the distal bioreactors. Additionally, tissues grown in autograft-filled bioreactors had higher compressive moduli and higher maximum screw pull-out forces than xenograft-filled bioreactors. In summary, we demonstrated that antibiotic-releasing space maintainers are an innovative approach to preserve a robust soft tissue pocket while clearing infection, and that local infections can increase local and remote bone growth.
Assuntos
Mandíbula , Reconstrução Mandibular , Animais , Antibacterianos/uso terapêutico , Reatores Biológicos , Porosidade , Próteses e Implantes , OvinosRESUMO
Cutaneous fungal infection is a challenging condition to treat that primarily afflicts immunocompromised patients. Local antifungal therapy may permit the delivery of high concentrations of antifungals directly to wounds while minimizing systemic toxicities. However, the field currently lacks suitable in vivo models. Therefore, a large cutaneous wound was created in immunosuppressed mice and inoculated with Aspergillus fumigatus. We fabricated biodegradable polymer microparticles (MPs) that were capable of locally delivering antifungal and characterized in vitro release kinetics. We compared wound bed size, fungal burden, and histological presence of fungi in mice treated with antifungal-loaded MPs. Mice with a cutaneous defect but no infection, mice with infected cutaneous defect but no treatment, and infected mice treated with blank MPs were used as controls. Infection of large wounds inhibited healing and resulted in tissue invasion in an inoculum-dependent manner. MPs were capable of releasing antifungals at concentrations above A. fumigatus Minimum Inhibitory Concentration (MIC) for at least 6 days. Wounds treated with MPs had significantly decreased size compared with no treatment (64.2% vs. 19.4% wound reduction, p = 0.002) and were not significantly different from uninfected controls (64.2% vs. 58.1%, p = 0.497). This murine model may serve to better understand cutaneous fungal infection and evaluate local biomaterials-based therapies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1867-1874, 2019.
Assuntos
Antifúngicos , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/metabolismo , Dermatomicoses/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Infecção dos Ferimentos/tratamento farmacológico , Animais , Antifúngicos/química , Antifúngicos/farmacocinética , Antifúngicos/farmacologia , Aspergilose/metabolismo , Aspergilose/patologia , Materiais Biocompatíveis/farmacocinética , Materiais Biocompatíveis/farmacologia , Dermatomicoses/metabolismo , Dermatomicoses/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecção dos Ferimentos/metabolismo , Infecção dos Ferimentos/patologiaRESUMO
As regenerative medicine matures as a field, more promising technologies are being translated from the benchtop to the clinic. However, many of these strategies are designed with otherwise healthy hosts in mind and validated in animal models without other co-morbidities. In reality, many of the patient populations benefiting from drug delivery and tissue engineering-based devices to enhance wound healing also have significant underlying immunodeficiency. Specifically, patients suffering from diabetes, malignancy, human immunodeficiency virus, post-organ transplantation, and other compromised states have significant pleotropic immune defects that affect wound healing. In this work, we review the role of different immune cells in the regenerative process, highlight the effect of several common immunocompromised states on wound healing, and discuss different drug delivery strategies for overcoming immunodeficiencies.