RESUMEN
Post-traumatic heterotopic ossification (HO) is the formation of ectopic bone in non-osseous structures following injury. The precise mechanism for bone development following trauma is unknown; however, early onset of HO may involve the production of pro-osteogenic serum factors. Here we evaluated serum from a cohort of civilian and military patients post trauma to determine early induction gene signatures in orthopaedic trauma induced HO. To test this, human adipose derived stromal/stem cells (hASCs) were stimulated with human serum from patients who developed HO following trauma and evaluated for a gene panel with qPCR. Pathway gene analysis ontology revealed that hASCs stimulated with serum from patients who developed HO had altered gene expression in the activator protein 1 (AP1) and AP1 transcriptional targets pathways. Notably, there was a significant repression in FOS gene expression in hASCs treated with serum from individuals with HO. Furthermore, the mitogen-activated protein kinase (MAPK) signaling pathway was activated in hASCs following serum exposure from individuals with HO. Serum from both military and civilian patients with trauma induced HO had elevated downstream genes associated with the MAPK pathways. Stimulation of hASCs with known regulators of osteogenesis (BMP2, IL6, Forskolin, and WNT3A) failed to recapitulate the gene signature observed in hASCs following serum stimulation, suggesting non-canonical mechanisms for gene regulation in trauma induced HO. These findings provide new insight for the development of HO and support ongoing work linking the systemic response to injury with wound specific outcomes.
Asunto(s)
Tejido Adiposo/citología , Sistema de Señalización de MAP Quinasas , Osificación Heterotópica/sangre , Osificación Heterotópica/etiología , Células Madre/enzimología , Heridas y Lesiones/complicaciones , Adulto , Diferenciación Celular , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Osteogénesis , Factor de Transcripción AP-1/metabolismo , Adulto JovenRESUMEN
Heterotopic ossification (HO) develops in the extremities of wounded service members and is common in the setting of high-energy penetrating injuries and blast-related amputations. No safe and effective prophylaxis modality has been identified for this patient population. Palovarotene has been shown to reduce bone formation in traumatic and genetic models of HO. The purpose of this study was to determine the effects of Palovarotene on inflammation, progenitor cell proliferation, and gene expression following a blast-related amputation in a rodent model (n = 72 animals), as well as the ability of Raman spectroscopy to detect early HO before radiographic changes are present. Treatment with Palovarotene was found to dampen the systemic inflammatory response including the cytokines IL-6 (p = 0.01), TNF-α (p = 0.001), and IFN-γ (p = 0.03) as well as the local inflammatory response via a 76% reduction in the cellular infiltration at post-operative day (POD)-7 (p = 0.03). Palovarotene decreased osteogenic connective tissue progenitor (CTP-O) colonies by as much as 98% both in vitro (p = 0.04) and in vivo (p = 0.01). Palovarotene treated animals exhibited significantly decreased expression of osteo- and chondrogenic genes by POD-7, including BMP4 (p = 0.02). Finally, Raman spectroscopy was able to detect differences between the two groups by POD-1 (p < 0.001). These results indicate that Palovarotene inhibits traumatic HO formation through multiple inter-related mechanisms including anti-inflammatory, anti-proliferative, and gene expression modulation. Further, that Raman spectroscopy is able to detect markers of early HO formation before it becomes radiographically evident, which could facilitate earlier diagnosis and treatment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1135-1144, 2018.
Asunto(s)
Células Madre Multipotentes/efectos de los fármacos , Osificación Heterotópica/prevención & control , Osteogénesis/efectos de los fármacos , Pirazoles/uso terapéutico , Estilbenos/uso terapéutico , Animales , Traumatismos por Explosión/complicaciones , Proliferación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Expresión Génica/efectos de los fármacos , Masculino , Osificación Heterotópica/etiología , Pirazoles/farmacología , Ratas Sprague-Dawley , Espectrometría Raman , Estilbenos/farmacología , Síndrome de Respuesta Inflamatoria Sistémica/prevención & control , Heridas Relacionadas con la Guerra/complicacionesRESUMEN
A pressing clinical need exists for 63% to 65% of combat-wounded service members and 11% to 20% of civilians who develop heterotopic ossification (HO) after blast-related extremity injury and traumatic injuries, respectively. The mammalian target of rapamycin pathway is a central cellular sensor of injury. We evaluated the prophylactic effects of rapamycin, a selective inhibitor of mammalian target of rapamycin signaling, on HO formation in a rat model of blast-related, polytraumatic extremity injury. Rapamycin was administered intraperitoneally daily for 14 days at 0.5 mg/kg or 2.5 mg/kg. Ectopic bone formation was monitored by micro-computed tomography and confirmed by histologic examination. Connective tissue progenitor cells, platelet-derived growth factor receptor-α-positive cells, and α-smooth muscle actin-positive blood vessels were assayed at postoperative day 7 by colony formation and immunofluorescence. Early gene expression changes were determined by low-density microarray. There was significant attenuation of 1) total new bone and soft tissue ectopic bone with 0.5 mg/kg (38.5% and 14.7%) and 2.5 mg/kg rapamycin (90.3% and 82.9%), respectively, 2) connective tissue progenitor cells, 3) platelet-derived growth factor receptor-α-positive cells, 4) α-smooth muscle actin-positive blood vessels, and 5) of key extracellular matrix remodeling (CD44, Col1a1, integrins), osteogenesis (Sp7, Runx2, Bmp2), inflammation (Cxcl5, 10, IL6, Ccl2), and angiogenesis (Angpt2) genes. No wound healing complications were noted. Our data demonstrate the efficacy of rapamycin in inhibiting blast trauma-induced HO by a multipronged mechanism.
Asunto(s)
Huesos/efectos de los fármacos , Osificación Heterotópica/prevención & control , Osteogénesis/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Traumatismos por Explosión/complicaciones , Huesos/patología , Modelos Animales de Enfermedad , Masculino , Osificación Heterotópica/patología , Osteogénesis/genética , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Microtomografía por Rayos X/métodosRESUMEN
Heterotopic ossification (HO) is a debilitating sequela of high-energy injuries. It frequently requires surgical excision once symptomatic and there is no practical prophylaxis for combat-injured patients. In this study, we examined the effect of local vancomycin powder on HO formation in a small animal model of blast-related, post-traumatic HO. Male Sprague-Dawley rats were subjected to a polytraumatic extremity injury and amputation with or without methicillin-resistant Staphylococcus aureus infection. Animals were randomized to receive a single local application of vancomycin (20 mg/kg) at the time of injury (POD-0, n = 34) or on postoperative day-3 (POD-3, n = 11). Quantitative volumetric measurement of ectopic bone was calculated at 12-weeks post-injury by micro-CT. Bone marrow and muscle tissues were also collected to determine the bacterial burden. Blood for serum cytokine analysis was collected at baseline and post-injury. Vancomycin treatment on POD-0 suppressed HO formation by 86% and prevented bone marrow and soft tissue infections. We concurrently observed a marked reduction histologically in nonviable tissue, chronic inflammatory cell infiltrates, bone infection, fibrous tissue, and areas of bone necrosis within this same cohort. Delayed treatment was significantly less efficacious. Neither treatment had a marked effect on the production of pro-inflammatory cytokines. Our study demonstrates that local vancomycin treatment at the time of injury significantly reduces HO formation in both the presence and absence of infection, with decreased efficacy if not given early. These findings further support the concept that the therapeutic window for prophylaxis is narrow, highlighting the need to develop early treatment strategies for clinical management. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2397-2406, 2017.
Asunto(s)
Antibacterianos/administración & dosificación , Osificación Heterotópica/prevención & control , Vancomicina/administración & dosificación , Heridas y Lesiones/complicaciones , Animales , Carga Bacteriana , Proliferación Celular/efectos de los fármacos , Citocinas/sangre , Evaluación Preclínica de Medicamentos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina , Osificación Heterotópica/sangre , Osificación Heterotópica/diagnóstico por imagen , Osificación Heterotópica/etiología , Ratas Sprague-Dawley , Infecciones de los Tejidos Blandos/etiología , Infecciones de los Tejidos Blandos/prevención & control , Infecciones Estafilocócicas/prevención & control , Síndrome de Respuesta Inflamatoria Sistémica/sangre , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Síndrome de Respuesta Inflamatoria Sistémica/prevención & control , Microtomografía por Rayos XRESUMEN
The pathologic development of heterotopic ossification (HO) is well described in patients with extensive trauma or with hyperactivating mutations of the bone morphogenetic protein (BMP) receptor ACVR1. However, identification of progenitor cells contributing to this process remains elusive. Here we show that connective tissue cells contribute to a substantial amount of HO anlagen caused by trauma using postnatal, tamoxifen-inducible, scleraxis-lineage restricted reporter mice (Scx-creERT2/tdTomatofl/fl ). When the scleraxis-lineage is restricted specifically to adults prior to injury marked cells contribute to each stage of the developing HO anlagen and coexpress markers of endochondral ossification (Osterix, SOX9). Furthermore, these adult preinjury restricted cells coexpressed mesenchymal stem cell markers including PDGFRα, Sca1, and S100A4 in HO. When constitutively active ACVR1 (caACVR1) was expressed in scx-cre cells in the absence of injury (Scx-cre/caACVR1fl/fl ), tendons and joints formed HO. Postnatal lineage-restricted, tamoxifen-inducible caACVR1 expression (Scx-creERT2/caACVR1fl/fl ) was sufficient to form HO after directed cardiotoxin-induced muscle injury. These findings suggest that cells expressing scleraxis within muscle or tendon contribute to HO in the setting of both trauma or hyperactive BMP receptor (e.g., caACVR1) activity. Stem Cells 2017;35:705-710.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linaje de la Célula , Músculos/patología , Osificación Heterotópica/patología , Tendones/patología , Receptores de Activinas Tipo I/metabolismo , Animales , Integrasas/metabolismo , Articulaciones/patología , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Osificación Heterotópica/etiología , Fenotipo , Heridas y Lesiones/complicaciones , Heridas y Lesiones/patologíaRESUMEN
Heterotopic ossification (HO) is the pathologic formation of bone separate from the normal skeleton. Although several models exist for studying HO, an understanding of the common in vitro properties of cells isolated from these models is lacking. We studied three separate animal models of HO including two models of trauma-induced HO and one model of genetic HO, and human HO specimens, to characterize the properties of cells derived from tissue containing pre-and mature ectopic bone in relation to analogous mesenchymal cell populations or osteoblasts obtained from normal muscle tissue. We found that when cultured in vitro, cells isolated from the trauma sites in two distinct models exhibited increased osteogenic differentiation when compared to cells isolated from uninjured controls. Furthermore, osteoblasts isolated from heterotopic bone in a genetic model of HO also exhibited increased osteogenic differentiation when compared with normal osteoblasts. Finally, osteoblasts derived from mature heterotopic bone obtained from human patients exhibited increased osteogenic differentiation when compared with normal bone from the same patients. These findings demonstrate that across models, cells derived from tissues forming heterotopic ossification exhibit increased osteogenic differentiation when compared with either normal tissues or osteoblasts. These cell types can be used in the future for in vitro investigations for drug screening purposes.
Asunto(s)
Huesos/citología , Quemaduras/complicaciones , Músculos/citología , Osificación Heterotópica/etiología , Osteoblastos/metabolismo , Adulto , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osificación Heterotópica/metabolismo , Osteoblastos/citología , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis , Ratas , Ratas Sprague-Dawley , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Factor de Transcripción Sp7 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Adulto JovenRESUMEN
Heterotopic ossification (HO) involves formation of endochondral bone at non-skeletal sites, is prevalent in severely wounded service members, and causes significant complications and delayed rehabilitation. As common prophylactic treatments such as anti-inflammatory drugs and irradiation cannot be used after multi-system combat trauma, there is an urgent need for new remedies. Previously, we showed that the retinoic acid receptor γ agonist Palovarotene inhibited subcutaneous and intramuscular HO in mice, but those models do not mimic complex combat injury. Thus, we tested Palovarotene in our validated rat trauma-induced HO model that involves blast-related limb injury, femoral fracture, quadriceps crush injury, amputation and infection with methicillin-resistant Staphylococcus aureus from combat wound infections. Palovarotene was given orally for 14days at 1mg/kg/day starting on post-operative day (POD) 1 or POD-5, and HO amount, wound dehiscence and related processes were monitored for up to 84days post injury. Compared to vehicle-control animals, Palovarotene significantly decreased HO by 50 to 60% regardless of when the treatment started and if infection was present. Histological analyses showed that Palovarotene reduced ectopic chondrogenesis, osteogenesis and angiogenesis forming at the injury site over time, while fibrotic tissue was often present in place of ectopic bone. Custom gene array data verified that while expression of key chondrogenic and osteogenic genes was decreased within soft tissues of residual limb in Palovarotene-treated rats, expression of cartilage catabolic genes was increased, including matrix metalloproteinase-9. Importantly, Palovarotene seemed to exert moderate inhibitory effects on wound healing, raising potential safety concerns related to dosing and timing. Our data show for the first time that Palovarotene significantly inhibits HO triggered by blast injury and associated complications, strongly indicating that it may prevent HO in patients at high risk such as those sustaining combat injuries and other forms of blast trauma.
Asunto(s)
Traumatismos por Explosión/tratamiento farmacológico , Osificación Heterotópica/tratamiento farmacológico , Receptores de Ácido Retinoico/metabolismo , Heridas y Lesiones/tratamiento farmacológico , Animales , Traumatismos por Explosión/complicaciones , Traumatismos por Explosión/patología , Condrogénesis/efectos de los fármacos , Condrogénesis/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Osificación Heterotópica/patología , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Pirazoles/farmacología , Pirazoles/uso terapéutico , Ratas Sprague-Dawley , Estilbenos/farmacología , Estilbenos/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/complicaciones , Heridas y Lesiones/patología , Receptor de Ácido Retinoico gammaRESUMEN
BACKGROUND: Heterotopic ossification (HO) affects the majority of combat-related lower extremity wounds involving severe fracture and amputation. Defining the timing of early osteogenic-related genes may help identify candidate prophylactic agents and guide the timing of prophylactic therapy after blast and other combat-related extremity injuries. QUESTIONS/PURPOSES: Using a recently developed animal model of combat-related HO, we sought to determine (1) the timing of early chondrogenesis, cartilage formation, and radiographic ectopic bone development; and (2) the early cartilage and bone-related gene and protein patterns in traumatized soft tissue. METHODS: We used an established rat HO model consisting of blast exposure, controlled femur fracture, crush injury, and transfemoral amputation through the zone of injury. Postoperatively, rats were euthanized on Days 3 to 28. We assessed evidence of early ectopic bone formation by micro-CT and histology and performed proteomic and gene expression analysis. RESULTS: All rats showed radiographic evidence of HO within 28 days. Key chondrogenic (collagen type I alpha 1 [COL1α1], p = 0.016) and osteogenic-related genes (Runt-related transcription factor 2 [RUNX-2], p = 0.029; osteoclacin [OCN], p = 0.032; phosphate-regulating neutral endopeptidase, X-linked [PHEX], p = 0.0290, and POU domain class 5 transcription factor [POU5F], p = 0.016) and proteins (Noggin [NOG], p = 0.04, OCN, p = 0.02, RUNX- 2, p = 0.04, and substance P-1 [SP-1], p = 0.01) in the injured soft tissue, normalized to the contralateral limb and/or sham-treated naïve rats, increased on Days 3 to 14 postinjury. By 14 days, foci of hypertrophic chondrocytes, hyaline cartilage, and woven bone were present in the soft tissue surrounding the amputation site. CONCLUSIONS: We found that genes that regulate early chondrogenic and osteogenic signaling and bone development (COL1α1, RUNX-2, OCN, PHEX, and POU5F1) are induced early during the tissue reparative/healing phase in a rat model simulating a combat-related extremity injury. CLINICAL RELEVANCE: The ability to correlate molecular events with histologic and morphologic changes will assist researchers and clinicians to understand HO and hence formulate therapeutic interventions.
Asunto(s)
Amputación Quirúrgica , Traumatismos por Explosión/complicaciones , Fracturas del Fémur/complicaciones , Osificación Heterotópica/etiología , Animales , Condrogénesis/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Marcadores Genéticos , Masculino , Osificación Heterotópica/diagnóstico por imagen , Osificación Heterotópica/genética , Osificación Heterotópica/metabolismo , Osificación Heterotópica/fisiopatología , Osteogénesis/genética , Ratas Sprague-Dawley , Transducción de Señal , Factores de Tiempo , Microtomografía por Rayos XRESUMEN
BACKGROUND: Heterotopic ossification (HO) develops in a majority of combat-related amputations wherein early bacterial colonization has been considered a potential early risk factor. Our group has recently developed a small animal model of trauma-induced HO that incorporates many of the multifaceted injury patterns of combat trauma in the absence of bacterial contamination and subsequent wound colonization. QUESTIONS/PURPOSES: We sought to determine if (1) the presence of bioburden (Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus [MRSA]) increases the magnitude of ectopic bone formation in traumatized muscle after amputation; and (2) what persistent effects bacterial contamination has on late microbial flora within the amputation site. METHODS: Using a blast-related HO model, we exposed 48 rats to blast overpressure, femur fracture, crush injury, and subsequent immediate transfemoral amputation through the zone of injury. Control injured rats (n = 8) were inoculated beneath the myodesis with phosphate-buffered saline not containing bacteria (vehicle) and treatment rats were inoculated with 1 × 10(6) colony-forming units of A baumannii (n = 20) or MRSA (n = 20). All animals formed HO. Heterotopic ossification was determined by quantitative volumetric measurements of ectopic bone at 12-weeks postinjury using micro-CT and qualitative histomorphometry for assessment of new bone formation in the residual limb. Bone marrow and muscle tissue biopsies were collected from the residual limb at 12 weeks to quantitatively measure the bioburden load and to qualitatively determine the species-level identification of the bacterial flora. RESULTS: At 12 weeks, we observed a greater volume of HO in rats infected with MRSA (68.9 ± 8.6 mm(3); 95% confidence interval [CI], 50.52-85.55) when compared with A baumannii (20.9 ± 3.7 mm(3); 95% CI, 13.61-28.14; p < 0.001) or vehicle (16.3 ± 3.2 mm(3); 95% CI, 10.06-22.47; p < 0.001). Soft tissue and marrow from the residual limb of rats inoculated with A baumannii tested negative for A baumannii infection but were positive for other strains of bacteria (1.33 × 10(2) ± 0.89 × 10(2); 95% CI, -0.42 × 10(2)-3.08 × 10(2) and 1.25 × 10(6) ± 0.69 × 10(6); 95% CI, -0.13 × 10(6)-2.60 × 10(6) colony-forming units in bone marrow and muscle tissue, respectively), whereas tissue from MRSA-infected rats contained MRSA only (4.84 × 10(1) ± 3.22 × 10(1); 95% CI, -1.47 × 10(1)-11.1 × 10(1) and 2.80 × 10(7) ± 1.73 × 10(7); 95% CI, -0.60 × 10(7)-6.20 × 10(7) in bone marrow and muscle tissue, respectively). CONCLUSIONS: Our findings demonstrate that persistent infection with MRSA results in a greater volume of ectopic bone formation, which may be the result of chronic soft tissue inflammation, and that early wound colonization may be a key risk factor. CLINICAL RELEVANCE: Interventions that mitigate wound contamination and inflammation (such as early débridement, systemic and local antibiotics) may also have a beneficial effect with regard to the mitigation of HO formation and should be evaluated with that potential in mind in future preclinical studies.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina/patogenicidad , Músculo Esquelético/microbiología , Osificación Heterotópica/microbiología , Osteogénesis , Infecciones Estafilocócicas/microbiología , Infección de Heridas/microbiología , Acinetobacter baumannii/patogenicidad , Amputación Quirúrgica , Animales , Carga Bacteriana , Biopsia , Traumatismos por Explosión/complicaciones , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Fracturas del Fémur/complicaciones , Masculino , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/lesiones , Músculo Esquelético/patología , Osificación Heterotópica/diagnóstico , Ratas Sprague-Dawley , Factores de Riesgo , Infecciones Estafilocócicas/diagnóstico , Factores de Tiempo , Infección de Heridas/diagnóstico , Microtomografía por Rayos XRESUMEN
Inducible systems providing temporal control of differentiation have the potential to improve outcomes in surgical reconstruction and regenerative medicine by precise modulation of wound healing and tissue repair processes. The aim of this study was to demonstrate that nanoformulated microRNA (miRNA) conjugates activated via photo exposure can lead to the induced osteogenic differentiation of human adipose-derived stromal/stem cells (hASCs) in vivo. The conjugate PC-miR-148b-SNP, a mimic of miRNA-148b tethered to silver nanoparticles (SNPs) via a photolabile linker, was used to modulate gene expression for improved closure of a critical size defect drilled on the right parietal bone of male CD-1 nude homozygous mice. The PC-miR-148b-SNP conjugates added to hASCs and loaded to either Matrigel or polycaprolactone (PCL) scaffolds resulted in different levels of healing of the defect. After 4 and 12weeks, 3-D micro-computed tomography reconstructed images indicate statistically significant defect closure from 3.83±1.19% to 5.46±2.01% and 6.54±4.28% to 32.53±8.3% for non-photoactivated and photoactivated conjugates, respectively, in the PCL scaffolds. The results were confirmed with H&E and Masson's Trichrome stains in the transverse sections of photoactivated conjugates. Collagen fiber staining was greatest at 12weeks when it reached approximately the same density and thickness as the native calvarium. This technology provides a platform that can be used with other miRNAs that actively govern the pathways responsible for regenerative and wound healing processes.
Asunto(s)
MicroARNs/fisiología , Nanopartículas , Fotoquímica , Cráneo/patología , Animales , Diferenciación Celular , Ratones , Microtomografía por Rayos XRESUMEN
The understanding that common broad-spectrum antimicrobials disrupt natural microbial flora important in acquiring nutrients and preventing infection has resulted in a paradigm shift favoring more selective antimicrobials. This work explores silver nanoparticles conjugated with ceragenin, or cationic antimicrobials (CSA-SNPs), as a potential Gram-positive selective antimicrobial. Herein, CSA-SNPs are characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, and high-performance liquid chromatography-electrospray time-of-flight mass spectrometry (HPLC-ESI-TOF-MS). The antimicrobial properties are determined through minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) and time-kill studies. Spatial selectivity of the conjugate nanoparticle was evaluated using confocal imaging, MATLAB statistical analysis, and video monitored interactions between bacteria and CSA-SNPs via laser trapping techniques. Cytotoxicity was also determined by live/dead staining and flow cytometry. Average particle size, as determined through TEM analysis, and hydrodynamic diameter, as determined via DLS, are 63.5 ± 38.8 and 102.23 ± 2.3 nm, respectively. The zeta potential of the SNP before and after CSA attachment is -18.23 and -8.34 mV, respectively. MIC/MBC data suggest that CSA-SNPs are 8 times more effective against Staphylococcus aureus than SNPs alone. Furthermore, MATLAB analysis of confocal imaging found that 70% of CSA-SNPs are within 2 µm of S. aureus, whereas this percentage falls to below 40% with respect to Escherichia coli. These results are bolstered further by laser trapping experiments demonstrating selective adherence of CSA-SNPs conjugates with bacterial strains. Cytotoxicity studies of CSA-SNPs against 3T3 fibroblasts indicate 50% cell viability at 50 ppm.
Asunto(s)
Antiinfecciosos/química , Nanopartículas del Metal/química , Plata/química , Esteroides/química , Antiinfecciosos/farmacología , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Staphylococcus aureus/efectos de los fármacosRESUMEN
Annually, more than 200,000 elective liposuction procedures are performed in the United States and over a million worldwide. The ease of harvest and abundance make human adipose-derived stromal/stem cells (hASCs) isolated from lipoaspirates an attractive, readily available source of adult stem cells that have become increasingly popular for use in many studies. Here, we describe common methods for hASC culture, preservation, and osteogenic differentiation. We introduce methods of ceramic, polymer, and composite scaffold synthesis with a description of morphological, chemical, and mechanical characterization techniques. Techniques for scaffold loading are compared, and methods for determining cell loading efficiency and proliferation are described. Finally, we provide both qualitative and quantitative techniques for in vitro assessment of hASC osteogenic differentiation.
Asunto(s)
Tejido Adiposo/citología , Células Madre/citología , Células del Estroma/citología , Andamios del Tejido/química , Adulto , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Separación Celular/métodos , Células Cultivadas , Cerámica/química , Criopreservación/métodos , Humanos , Osteogénesis , Polímeros/químicaRESUMEN
Nationally, nearly 1.5 million patients in the USA suffer from ailments requiring bone grafts and hip and other joint replacements. Infections following internal fixation in orthopaedic trauma can cause osteomyelitis in 22-66% of cases and, if uncontrolled, the mortality rate can be as high as 2%. We characterize a procedure for the synthesis of antimicrobial and biocompatible poly-l-lactic acid (PLLA) and poly-ethyleneglycol (PEG) bioscaffolds designed to degrade and absorb at a controlled rate. The bioscaffold architecture aims to provide a suitable substrate for the controlled release of silver nanoparticles (SNPs) to reduce bacterial growth and to aid the proliferation of human adipose-derived stem cells (hASCs) for tissue-engineering applications. The fabricated bioscaffolds were characterized by scanning transmission microscope (SEM) and it showed that the addition of tncreasing concentrations of SNPs results in the formation of dendritic porous channels perpendicular to the axis of precipitation. The antimicrobial properties of these porous bioscaffolds were tested according to a modified ISO 22196 standard across varying concentrations of biomass-mediated SNPs to determine an efficacious antimicrobial concentration. The bioscaffolds reduced the Staphylococcus aureus and Escherichia coli viable colony-forming units by 98.85% and 99.9%, respectively, at an antimicrobial SNPs concentration of 2000 ppm. Human ASCs were seeded on bioscaffolds and cultured in vitro for 20 days to study the effect of SNPs concentration on the viability of cells. SEM analysis and the metabolic activity-based fluorescent dye, AlamarBlue®, demonstrated the growth of cells on the efficacious antimicrobial bioscaffolds. The biocompatibility of in vitro leached silver, quantified by inductively coupled plasma optical emission spectroscopy (ICP-OES), proved non-cytotoxic when tested against hASCs, as evaluated by MTT assay.
Asunto(s)
Antiinfecciosos/farmacología , Materiales Biocompatibles/farmacología , Ortopedia , Prótesis e Implantes , Andamios del Tejido/química , Tejido Adiposo/citología , Muerte Celular/efectos de los fármacos , Fuerza Compresiva/efectos de los fármacos , Módulo de Elasticidad/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Humanos , Ácido Láctico/farmacología , Nanopartículas del Metal/ultraestructura , Pruebas de Sensibilidad Microbiana , Poliésteres , Polietilenglicoles/farmacología , Polímeros/farmacología , Plata/farmacología , Staphylococcus aureus/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células Madre/ultraestructuraRESUMEN
Herein we report a facile layer-by-layer method for creating an antimicrobial coating composed of silver nanoparticles on medical grade titanium test discs. Nanoscale silver nanoparticle layers are attached to the titanium orthopedic implant material via aminopropyltriethoxy silane crosslinker that reacts with neighboring silane moieties to create an interconnected network. A monolayer of silane, followed by a monolayer of silver nanoparticles would form one self-assembled layer and this process can be repeated serially, resulting in increased silver nanoparticles deposition. The release rate of silver ion increases predictably with increasing numbers of layers and at appropriate thicknesses these coatings demonstrate 3-4 log reduction of viable Escherichia coli and Staphylococcus aureus bacteria. Increasing the thickness of the coatings resulted in reduced bacterial colonization as determined by fluorescent staining and image analysis. Interestingly, the cytotoxicity of murine 3T3 cells as quantified by fluorescent staining and flow cytometry, was minimal and did not vary significantly with the coating thickness. Additionally, these coatings are mechanically stable and resist delamination by orthogonal stress test. This simple layer-by-layer coating technique may provide a cost-effective and biocompatible method for reducing microbial colonization of implantable orthopedic devices.
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Infecciones Bacterianas/prevención & control , Supervivencia Celular , Materiales Biocompatibles Revestidos , Nanopartículas del Metal/química , Plata/química , Células 3T3 , Animales , Adhesión Bacteriana , Infecciones Bacterianas/microbiología , Escherichia coli/aislamiento & purificación , Técnicas In Vitro , Ratones , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Espectrofotometría Ultravioleta , Staphylococcus aureus/aislamiento & purificación , Resonancia por Plasmón de Superficie , Resistencia a la TracciónRESUMEN
New synthesis techniques are providing increasing control over many inorganic nanoparticle characteristics, facilitating the creation of new multifunctional theranostics. This report proposes the synthesis and testing of a combination nanoparticle comprised of a maghemite core for enhanced T2 MRI contrast diagnostics, a colloidal silver shell acting as an antimicrobial and therapeutic vehicle, and a ceragenin (CSA-124) surfactant providing microbial adhesion. A polyacrylic acid functionalized maghemite nanoparticle is synthesized by a high temperature organic phase reduction followed by thiol functionalization and gold cluster seeding. A silver shell is formed through AgNO3 reduction, and an oriented monolayer of the thiolated ceragenin, is bound through a self-assembly process. The process and products are characterized throughout synthesis through TEM, DLS, FT-IR, UV-Vis, ICP-OES, HPLC-ESI-TOF-MS, DC magnetization and susceptibility, X-ray diffraction, and in vitro MRI. Synthesized Diagnostic Antimicrobial Nanoparticles (DANs) were found to have a spherical morphology with a diameter of 32.47±1.83 nm, hydrodynamic diameter of 53.05±1.20 nm, maximum magnetic moment of 12 emu/g NP (54 emu/g Fe) with little variation due to temperature, and are predominantly paramagnetic. In vitro MRI studies show that DANs contrast well at concentrations as low as 9 ppm, and successfully adhere to Staphylococcus aureus. DAN MIC was determined to be approximately 12 ppm and 24 ppm against S. aureus and Escherichia coli respectively.
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Bacterias/química , Compuestos Férricos/química , Plata/química , Esteroides/química , Cromatografía Líquida de Alta Presión , Microscopía Electrónica de Transmisión , Espectrometría de Masa por Ionización de Electrospray , Difracción de Rayos XRESUMEN
Delivery systems providing spatial and temporal control have the potential to improve outcomes in surgical reconstruction and regenerative medicine by precise modulation of wound healing and tissue repair processes. In this study we describe a synthesis and oligonucleotide functionalization process of silver nanoparticle complexes for photoactivated microRNA (miRNA) delivery. The activity of the PC-miR-148b-SNP construct is demonstrated by light mediated delivery of miR-148b mimic resulting in differentiation of human autologous adipose derived mesenchymal stromal/stem cells (hASCs) into an osteogenic linage. The conjugate, upon photoactivation, increases alkaline phosphatase (ALP) activity in the cell membrane and calcification (mineralization) of hASCs on days 7 and 14 respectively. Additionally, the expression of mRNA for the early, middle and late stage osteogenic markers; ALP, RunX2 and osteocalcin (OCN) respectively, was also significantly upregulated at days 7 and 28, respectively after photoactivation of PC-miR-148b-SNP and release of miR-148b mimics. Additionally, PC-miR-148b-SNP conjugate is readily delivered to the intracellular compartment without the use of transfection vectors commonly required for free oligonucleotides. This technology demonstrates photo-controlled, spatial and temporal modulation of osteogenesis in hASCs.
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Adipocitos/citología , Luz , Nanopartículas del Metal/química , MicroARNs/química , Osteogénesis/efectos de los fármacos , Células del Estroma/citología , Ingeniería de Tejidos/métodos , Adulto , Células Cultivadas , Femenino , Humanos , Persona de Mediana Edad , Plata/química , Células del Estroma/efectos de los fármacos , Adulto JovenRESUMEN
Electrospun fibrous bio-nanocomposite scaffolds reinforced with cellulose nanocrystals (CNCs) were fabricated by using maleic anhydride (MAH) grafted poly(lactic acid) (PLA) as matrix with improved interfacial adhesion between the two components. Morphological, thermal, mechanical, and in vitro degradation properties as well as basic cytocompatibility using human adult adipose derived mesenchymal stem cells (hASCs) of MAH grafted PLA/CNC (i.e., MPLA/CNC) scaffolds were characterized. Morphological investigation indicated that the diameter and polydispersity of electrospun MPLA/CNC nanofibers were reduced with the increased CNC content. The addition of CNCs improved both the thermal stability and mechanical properties of MPLA/CNC composites. The MPLA/CNC scaffolds at the 5 wt % CNC loading level showed not only superior tensile strength (more than 10 MPa), but also improved stability during in vitro degradation compared with the MPLA and PLA/CNC counterparts. Moreover, the fibrous MPLA/CNC composite scaffolds were non-toxic to hASCs and capable of supporting cell proliferation. This study demonstrates that fibrous MPLA/CNC bio-nanocomposite scaffolds are biodegradable, cytocompatible, and possess useful mechanical properties for bone tissue engineering.
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Celulosa/química , Técnicas Electroquímicas/métodos , Ácido Láctico/química , Nanocompuestos/química , Nanopartículas/química , Polímeros/química , Andamios del Tejido/química , Supervivencia Celular/fisiología , Células Cultivadas , Humanos , Anhídridos Maleicos/química , Ensayo de Materiales , Células Madre Mesenquimatosas/citología , Nanocompuestos/ultraestructura , Nanotecnología/métodos , Poliésteres , Temperatura , Ingeniería de TejidosRESUMEN
The unique photophysical properties of noble metal nanoparticles contribute to their potential as photoactivated drug delivery vectors. Here we demonstrate the synthesis and characterization of 60-80 nm silver nanoparticles (SNPs) decorated with thiol-terminated photolabile DNA oligonucleotides. In vitro assays and fluorescent confocal microscopy of treated cell cultures show efficient UV-wavelength photoactivation of surface-tethered caged ISIS2302 antisense oligonucleotides possessing internal photocleavable linkers. As a demonstration of the advantages of these novel nanocarriers, we investigate properties including: enhanced stability to nucleases, increased hybridization activity upon photorelease, and efficient cellular uptake as compared to commercial transfection vectors. Their potential as multicomponent delivery agents for oligonucleotide therapeutics is shown through regulation of ICAM-1 (Intracellular Adhesion Molecule-1) silencing. Our results suggest a means to achieve light-triggered, spatiotemporally controlled gene silencing via nontoxic silver nanocarriers, which hold promise as tailorable platforms for nanomedicine, gene expression studies, and genetic therapies.