RESUMEN
Implantation of left ventricular assist devices typically requires cardiopulmonary bypass support, which is associated with postoperative complications. A novel suture-less inflow cannula, which can be implanted without bypass, uses mild myocardial compression to seal the interface, however, this may lead to necrosis of the myocardium. To circumvent this issue, a bilayered scaffold has been developed to promote tissue growth at the interface between cannula and myocardium. The bilayered scaffold consists of a silicone base layer, which mimics the seal, and a melt electrospun polycaprolactone scaffold to serve as a tissue integration layer. Biocompatibility of the bilayered scaffolds was assessed by analyzing cell viability, morphology, and metabolic activity of human foreskin fibroblasts cultured on the scaffolds for up to 14 days. There was no evidence of cytotoxicity and the cells adhered readily to the bilayered scaffolds, revealing a cell morphology characteristic of fibroblasts, in contrast to the low cell adhesion observed on flat silicone sheets. The rate of cell proliferation on the bilayered scaffolds rose over the 14-day period and was significantly greater than cells seeded on the silicone sheets. This study suggests that melt electrospun bilayered scaffolds have the potential to support tissue integration of a suture-less inflow cannula for cardiovascular applications. Furthermore, the method of fabrication described here and the application of bilayered scaffolds could also have potential uses in a diverse range of biomedical applications.
Asunto(s)
Cánula , Cateterismo/instrumentación , Corazón Auxiliar , Poliésteres/química , Andamios del Tejido/química , Adhesión Celular , Línea Celular , Supervivencia Celular , Diseño de Equipo , Fibroblastos/citología , Humanos , Silicio/química , Suturas , Ingeniería de Tejidos/métodosRESUMEN
Polycaprolactone with five different molecular weights was spin-coated on silicon wafers and plasma immersion ion implanted (PIII) with ion fluence in the range 5 × 1014-2 × 1016 ions/cm2. The effects of PIII treatment on the optical properties, chemical structure, crystallinity, morphology, gel fraction formation and wettability were investigated. As in the case of a number of previously studied polymers, oxidation and hydrophobic recovery of the PIII treated PCL follow second order kinetics. CAPA 6250, which has the lowest molecular weight and the highest degree of crystallinity of the untreated PCL films studied, has the highest carbonization of the modified layer after PIII treatment. Untreated medical grade PCL films, mPCL PC12 (Perstorp) and mPCL OsteoporeTM have similar chemical structures and crystallinity. Accordingly, the chemical and structural transformations caused by PIII treatment and post-treatment oxidation are almost identical for these two polymers. In general, PIII treatment destroys the nano-scale lamellar structure and results in a reduction of PCL crystallinity. Examination after washing PIII treated PCL films in toluene confirmed our hypothesis that cross-linking due to PIII treatment is significantly higher in semi-crystalline PCL as compared with amorphous polymers.
Asunto(s)
Materiales Biocompatibles Revestidos/química , Iones/química , Poliésteres/química , Propiedades de Superficie , Carbono/química , Cristalización , Ensayo de Materiales , Microscopía de Fuerza Atómica , Peso Molecular , Oxígeno/química , Plasma , Polímeros/química , Proteínas/química , Refractometría , Silicio/química , Espectroscopía Infrarroja por Transformada de Fourier , Humectabilidad , Difracción de Rayos XRESUMEN
Lycopene, a compound that blocks the action of free radicals and oxygen molecules, is found in tomatoes and tomato-based products and linked to a reduced incidence of cancer. Increasing willingness of patients to maintain a healthy lifestyle by supplemental intake of nutrients and acceptance of alternative therapeutics has boosted research into nutraceuticals. The potential of lycopene to prevent or treat cancer has been investigated, but outcomes are inconsistent and its mode of action is still unknown. Further studies are needed to understand the role of lycopene in cancer prevention and treatment. The impact of lycopene on viability, proliferation, migration, and invasion of five different cancer cell lines was determined using monolayer and spheroid cultures. Cell viability was significantly reduced upon lycopene treatment at physiologically attainable concentrations. Cell proliferation, migration, and invasion did not change upon lycopene treatment. Ovarian cancer spheroids initially showed a decreased proliferation and after 14 days increased cell viability upon lycopene treatment, confirming the potential of lycopene to reduce cancer cell growth in short-term cultures and also indicate enhanced cell viability over prolonged exposure. This study cannot substantiate that lycopene inhibits cell functions associated with tumor growth, even in a 3D cancer model that mimics the natural tumor microenvironment.
Asunto(s)
Anticarcinógenos/farmacología , Carotenoides/farmacología , Técnicas de Cultivo de Célula/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Licopeno , Masculino , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
Chlamydia pneumoniae is a common human and animal pathogen associated with upper and lower respiratory tract infections. Of the animal C. pneumoniae isolates, the koala nasal isolate (LPCoLN) is by far the best genetically characterised. This current study was designed to characterise the morphology and developmental events for the LPCoLN isolate, and our results showed several striking in vitro growth differences when compared to the human isolate, AR39. The LPCoLN inclusion size and morphology was distinct from AR39, and a much faster doubling time (3.4-4.9h versus 5.9-8.7h doubling time) was observed when grown in HEp-2 cell monolayers. Confocal and electron microscopy of LPCoLN confirmed large (9-30 microm in diameter) inclusions, that were heterogeneously shaped, compared to the small (5-9 microm in diameter), uniformly shaped inclusions of AR39. The morphology of the LPCoLN elementary body was round, and had a narrow or nonexistent periplasmic space, compared to the 'pear-shaped' morphology of AR39 EBs. While both isolates showed evidence of inclusion fusion, the level of fusion was much higher for LPCoLN (100%) compared to AR39 (30-40%). Our findings have provided new insights and identified key differences in the in vitro doubling time, size and morphology of an animal C. pneumoniae isolate.
Asunto(s)
Infecciones por Chlamydophila/veterinaria , Chlamydophila pneumoniae/fisiología , Phascolarctidae/microbiología , Infecciones del Sistema Respiratorio/veterinaria , Animales , Infecciones por Chlamydophila/microbiología , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/crecimiento & desarrollo , Chlamydophila pneumoniae/ultraestructura , ADN Bacteriano/química , ADN Bacteriano/genética , Dosificación de Gen , Microscopía Confocal/veterinaria , Microscopía Electrónica de Transmisión/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , Infecciones del Sistema Respiratorio/microbiologíaRESUMEN
Medication-related osteonecrosis of the jaw (MRONJ) poses an ongoing challenge for clinicians and researchers. Currently, there is a lack of preventative measures available for at-risk patients undergoing tooth extractions, especially those with prior bisphosphonate treatment due to osteoporosis or bone metastasis diagnoses. Here, these issues are addressed using a preventative tissue engineering strategy against MRONJ development. This study evaluates the efficacy of a poly(ethylene glycol)-heparin hydrogel as a tool for the delivery of arginylglycylaspartic acid (RGD) and recombinant human bone morphogenic protein-2 (rhBMP-2). Three groups of skeletally mature rats each receive two doses of intravenous zoledronic acid prior to surgery and undergo extraction of the right first mandibular molar with gingival closure. Experimental groups either have the sockets left empty, filled with hydrogel minus rhBMP-2, or filled with hydrogel plus rhBMP-2. Eight weeks postoperatively specimens are analyzed using radiological, histological, and scanning electron microscopy (SEM) techniques. µCT analysis shows increased bone formation with hydrogel/rhBMP-2 delivery compared to the empty socket. Hydrogel-treated groups display increased presence of osteocytes and increased osteoclastic action compared to the empty sockets. These results represent the first step toward improved delivery of rhBMP-2 and a potential MRONJ preventative for patients undergoing bisphosphonate treatment.
Asunto(s)
Osteonecrosis de los Maxilares Asociada a Difosfonatos/prevención & control , Proteína Morfogenética Ósea 2/farmacocinética , Preparaciones de Acción Retardada/farmacocinética , Liberación de Fármacos , Factor de Crecimiento Transformador beta/farmacocinética , Animales , Osteonecrosis de los Maxilares Asociada a Difosfonatos/patología , Proteína Morfogenética Ósea 2/administración & dosificación , Células Cultivadas , Quimioprevención/métodos , Preparaciones de Acción Retardada/administración & dosificación , Modelos Animales de Enfermedad , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Humanos , Hidrogeles/farmacocinética , Osteocitos/efectos de los fármacos , Osteocitos/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Factor de Crecimiento Transformador beta/administración & dosificaciónRESUMEN
Representative in vitro models that mimic the native bone tumor microenvironment are warranted to support the development of more successful treatments for bone metastases. Here, we have developed a primary cell 3D model consisting of a human osteoblast-derived tissue-engineered construct (hOTEC) indirectly co-cultured with patient-derived prostate cancer xenografts (PDXs), in order to study molecular interactions in a patient-derived microenvironment context. The engineered biomimetic microenvironment had high mineralization and embedded osteocytes, and supported a high degree of cancer cell osteomimicry at the gene, protein and mineralization levels when co-cultured with prostate cancer PDXs from a lymph node metastasis (LuCaP35) and bone metastasis (BM18) from patients with primary prostate cancer. This fully patient-derived model is a promising tool for the assessment of new molecular mechanisms and as a personalized pre-clinical platform for therapy testing for patients with prostate cancer bone metastases.
Asunto(s)
Biomimética , Neoplasias Óseas/secundario , Osteoblastos/patología , Neoplasias de la Próstata/patología , Ingeniería de Tejidos , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Anciano , Animales , Matriz Ósea/metabolismo , Neoplasias Óseas/genética , Huesos/patología , Huesos/ultraestructura , Calcificación Fisiológica , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Matriz Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones Endogámicos NOD , Osteocitos/metabolismo , Osteocitos/ultraestructura , Andamios del Tejido/químicaRESUMEN
BACKGROUND: Chlamydia trachomatis, an obligate intracellular human pathogen, is the most prevalent bacterial sexually transmitted infection worldwide and a leading cause of preventable blindness. HtrA is a virulence and stress response periplasmic serine protease and molecular chaperone found in many bacteria. Recombinant purified C. trachomatis HtrA has been previously shown to have both activities. This investigation examined the physiological role of Chlamydia trachomatis HtrA. RESULTS: The Chlamydia trachomatis htrA gene complemented the lethal high temperature phenotype of Escherichia coli htrA- (>42 degrees C). HtrA levels were detected to increase by western blot and immunofluorescence during Chlamydia heat shock experiments. Confocal laser scanning microscopy revealed a likely periplasmic localisation of HtrA. During penicillin induced persistence of Chlamydia trachomatis, HtrA levels (as a ratio of LPS) were initially less than control acute cultures (20 h post infection) but increased to more than acute cultures at 44 h post infection. This was unlike IFN-gamma persistence where lower levels of HtrA were observed, suggesting Chlamydia trachomatis IFN-gamma persistence does not involve a broad stress response. CONCLUSION: The heterologous heat shock protection for Escherichia coli, and increased HtrA during cell wall disruption via penicillin and heat shock, indicates an important role for HtrA during high protein stress conditions for Chlamydia trachomatis.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/biosíntesis , Chlamydia trachomatis/fisiología , Respuesta al Choque Térmico , Interferón gamma/farmacología , Penicilinas/farmacología , Serina Endopeptidasas/biosíntesis , Western Blotting , Línea Celular , Chlamydia trachomatis/efectos de los fármacos , Chlamydia trachomatis/efectos de la radiación , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Prueba de Complementación Genética , Humanos , Microscopía Fluorescente , Chaperonas Moleculares/biosíntesis , Periplasma/químicaRESUMEN
OBJECTIVE: The objective of the study was to determine the effects in late gestation of Ureaplasma parvum serovar 3 colonization and the effects, preterm, of U. parvum serovar 6. STUDY DESIGN: Ewes received an intraamniotic (i.a.) injection of U. parvum serovar 6 (20 x 10(6) colony-forming units [cfu]; n = 9), U. parvum serovar 3 (20 x 10(3) cfu; n = 6), vehicle (n = 10), or saline (n = 4) on day 80 of pregnancy (d). The lambs were delivered at 125 d (U. parvum serovar 6, n = 9; saline or media controls, n = 9) or 145 d (U. parvum serovar 3, n = 6; media controls, n = 5) for assessment of inflammation and lung maturation. RESULTS: I.a. ureaplasmas caused histologic chorioamnionitis but not preterm delivery. Fetal lung epithelium was colonized with ureaplasmas at both gestational ages, and pulmonary interleukin-8 levels had doubled in the ureaplasma-colonized animals, compared with the controls at 145 d. Surfactant levels in bronchoalveolar lavage fluid had increased 8-fold and 2.5-fold at 125 and 145 d, respectively, after ureaplasma injection. CONCLUSION: Fetal lung inflammation and altered development accompanies ureaplasma colonization, regardless of age at delivery.
Asunto(s)
Corioamnionitis/fisiopatología , Preñez , Infecciones por Ureaplasma , Ureaplasma/patogenicidad , Líquido Amniótico/microbiología , Animales , Animales Recién Nacidos , Corioamnionitis/microbiología , Modelos Animales de Enfermedad , Femenino , Madurez de los Órganos Fetales , Embarazo , Distribución Aleatoria , Valores de Referencia , Oveja DomésticaRESUMEN
Modern tissue engineering technologies have delivered tools to recreate a cell's naturally occurring niche in vitro and to investigate normal and pathological cell-cell and cell-niche interactions. Hydrogel biomaterials mimic crucial properties of native extracellular matrices, including mechanical support, cell adhesion sites and proteolytic degradability. As such, they are applied as 3D cell culture platforms to replicate tissue-like architectures observed in vivo, allowing physiologically relevant cell behaviors. Here we review bioengineered 3D approaches used for prostate and breast cancer. Furthermore, we describe the synthesis and use of gelatin methacryloyl-based hydrogels as in vitro 3D cancer model. This platform is used to engineer the microenvironments for prostate and breast cancer cells to study processes regulating spheroid formation, cell functions and responses to therapeutic compounds. Collectively, these bioengineered 3D approaches provide cell biologists with innovative pre-clinical tools that integrate the complexity of the disease seen in patients to advance our knowledge of cancer cell physiology and the contribution of a tumor's surrounding milieu.
Asunto(s)
Neoplasias de la Mama/patología , Gelatina , Hidrogeles , Neoplasias de la Próstata/patología , Esferoides Celulares , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Femenino , Gelatina/química , Humanos , Hidrogeles/química , Masculino , Metacrilatos/química , Células Tumorales CultivadasRESUMEN
OBJECTIVE: There is a paucity of data characterizing the microbiota of the female upper genital tract, which controversially is described as a sterile site. We examine whether the fallopian tube harbours an endogenous microbial community. DESIGN: This prospective study collected from women undergoing total hysterectomy or salpingectomy-oophorectomy. SETTING: Private hospital gynaecology department. PATIENTS: Fallopian tubes were collected from women diagnosed with benign disease or for prophylaxis. INTERVENTIONS: Samples were interrogated for the presence of microbial DNA using a next generation sequencing technology approach to exploit the V5 to V9 regions of the 16S rRNA gene. MAIN OUTCOME MEASURES: The fallopian tube microbiota was characterized using traditional culture techniques and next generation sequencing. RESULTS: Bacteria were isolated from 50% of cultured samples, and 100% of samples returned positive PCR results. Only 68% of the culture isolates could be confidently identified using automated diagnostic equipment in a clinical microbiology laboratory. Monomicrobial communities were identified only for cultured isolates (50%). Pyrosequencing revealed that all communities were polymicrobial. Lactobacillus spp. were not present in all groups, nor were they the most dominant isolates. Distinct differences in the microbial communities were evident for left compared to right fallopian tubes, ampulla versus isthmus, pre- and post- menopausal tissue, and in secretory phase fallopian tubes with and without Mirena intrauterine devices in situ (all p < 0.05). CONCLUSION: The female upper genital tract is not sterile. Distinct microbial community profiles in the fallopian tubes of healthy women suggest that this genital tract site supports an endogenous microbiota.
RESUMEN
Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell® ). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+ /K+ -ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Lámina Basal de la Coroides , Proteínas del Ojo/biosíntesis , Regulación de la Expresión Génica , Ensayo de Materiales , Membranas Artificiales , Epitelio Pigmentado de la Retina/metabolismo , Línea Celular , Fibroínas , Humanos , Degeneración Macular/metabolismo , Degeneración Macular/terapia , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Selection of decalcification agents is an essential consideration when processing mineralized tissues because the integrity and immunohistochemical characteristics of the tissues may be affected. Here, we report results obtained from the decalcification of rat mandibles using 10% ethylenediaminetetraacetic acid (EDTA) at room temperature (RT), 10% EDTA at 37C, 5% nitric acid, and 10% formic acid at RT. Decalcification endpoints were determined by microcomputed tomography. Morphological preservation and antigenicity were evaluated by hematoxylin and eosin staining and immunohistochemistry. Decalcification of the anterior and posterior portions of the mandible took 220 and 191 hr in 10% EDTA RT, 102 and 73 hr in 10% EDTA 37C, 13.5 and 4.3 hr in 5% nitric acid, and 140 and 36 hr in 10% formic acid, respectively. Decalcification in 10% EDTA at 37C was accelerated, but 10% EDTA at RT provided optimal results for immunohistochemistry and cellular and structural details. Decalcification using 5% nitric acid was accomplished in the shortest time and exhibited good cellular and architectural morphology, whereas 10% formic acid was suboptimal with respect to tissue and cellular morphology. Despite being the slowest method, EDTA at RT is still the recommended method for decalcifying mineralized tissues; however, if rapid decalcification is needed, 5% nitric acid is the best option, yielding acceptable tissue integrity and speed.
Asunto(s)
Técnica de Descalcificación/métodos , Mandíbula/citología , Animales , Antígenos/metabolismo , Mandíbula/inmunología , Mandíbula/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Various in vitro culture systems have been used to investigate the pathogenesis of age-related macular degeneration (AMD). However, many still rely on oversimplified monolayer culture models. AMD is a complex disease, associated with the pathological changes to multiple structural components such as the Bruch's membrane, retinal pigment epithelium (RPE), and choroidal endothelial cells. This study aims to construct a novel 3D coculture model using the polycaprolactone (PCL)-gelatin electrospun scaffold, with human RPE cells (hRPE) and primate choroidal cells (RF-6A). Results from this study show that PCL-gelatin scaffolds have a highly porous ultrastructure that supports the attachment, proliferation, differentiation, and migration of the hRPEs and choroidal endothelial cells. It is also demonstrated that the PCL-gelatin 3D coculture model may be useful in exploring the molecular interplay between the hPRE and the choroidal endothelial cells, and their effects on growth factor modulation, which may be important in the pathogenesis of AMD.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/patología , Animales , Técnicas de Cultivo de Célula/instrumentación , Coroides/citología , Ensayo de Inmunoadsorción Enzimática , Proteínas del Ojo/metabolismo , Gelatina/química , Haplorrinos , Humanos , Membranas Artificiales , Microscopía Electrónica de Rastreo , Factores de Crecimiento Nervioso/metabolismo , Fagocitosis , Poliésteres/química , Epitelio Pigmentado de la Retina/citología , Serpinas/metabolismo , Andamios del Tejido , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We set out to compare the osteogenicity of human mesenchymal stem (hMSCs) and osteoblasts (hOBs). Upon osteogenic induction in monolayer, hMSCs showed superior matrix mineralization expressing characteristic bone-related genes. For scaffold cultures, both cell types presented spindle-shaped, osteoblast-like morphologies forming a dense, interconnected network of high viability. On the scaffolds, hOBs proliferated faster. A general upregulation of parathyroid hormone-related protein (PTHrP), osteoprotegrin (OPG), receptor activator of NF-κB ligand (RANKL), sclerostin (SOST), and dentin matrix protein 1 (DMP1) was observed for both cell types. Simultaneously, PTHrP, RANKL and DMP-1 expression decreased under osteogenic stimulation, while OPG and SOST increased significantly. Following transplantation into NOD/SCID mice, µCT and histology showed increased bone deposition with hOBs. The bone was vascularized, and amounts further increased for both cell types after recombinant human bone morphogenic protein 7 (rhBMP-7) addition also stimulating osteoclastogenesis. Complete bone organogenesis was evidenced by the presence of osteocytes and hematopoietic precursors. Our study results support the asking to develop 3D cellular models closely mimicking the functions of living tissues suitable for in vivo translation.
Asunto(s)
Resorción Ósea/fisiopatología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteogénesis/fisiología , Proteína Relacionada con la Hormona Paratiroidea/química , Animales , Resorción Ósea/metabolismo , Humanos , Ratones , Ratones SCID , Osteoblastos/química , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/fisiologíaRESUMEN
The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine.
Asunto(s)
Materiales Biocompatibles , Ingeniería de Tejidos , Reactores Biológicos , Técnicas de Cultivo de Célula , Humanos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Osteoblastos/citologíaRESUMEN
The use of biopolymers as a three dimensional (3D) support structure for cell growth is a leading tissue engineering approach in regenerative medicine. Achieving consistent cell seeding and uniform cell distribution throughout 3D scaffold culture in vitro is an ongoing challenge. Traditionally, 3D scaffolds are cultured within tissue culture plates to enable reproducible cell seeding and ease of culture media change. In this study, we compared two different well-plates with different surface properties to assess whether seeding efficiencies and cell growth on 3D scaffolds were affected. Cell attachment and growth of murine calvarial osteoblast (MC3T3-E1) cells within a melt-electrospun poly-ε-caprolactone scaffold were assessed when cultured in either "low-adhesive" non-treated or corona discharged-treated well-plates. Increased cell adhesion was observed on the scaffold placed in the surface treated culture plates compared to the scaffold in the non-treated plates 24 h after seeding, although it was not significant. However, higher cell metabolic activity was observed on the bases of all well-plates than on the scaffold, except for day 21, well metabolic activity was higher in the scaffold contained in non-treated plate than the base. These results indicate that there is no advantage in using non-treated plates to improve initial cell seeding in 3D polymeric tissue engineering scaffolds, however non-treated plates may provide an improved metabolic environment for long-term studies.
RESUMEN
Bone metastasis is a frequent and life-threatening complication of breast cancer. The molecular mechanisms supporting the establishment of breast cancer cells in the skeleton are still not fully understood, which may be attributed to the lack of suitable models that interrogate interactions between human breast cancer cells and the bone microenvironment. Although it is well-known that integrins mediate adhesion of malignant cells to bone extracellular matrix, their role during bone colonization remains unclear. Here, the role of ß1 integrins in bone colonization was investigated using tissue-engineered humanized in vitro and in vivo bone models. In vitro, bone-metastatic breast cancer cells with suppressed integrin ß1 expression showed reduced attachment, spreading, and migration within human bone matrix compared to control cells. Cell proliferation in vitro was not affected by ß1 integrin knockdown, yet tumor growth in vivo within humanized bone microenvironments was significantly inhibited upon ß1 integrin suppression, as revealed by quantitative in/ex vivo fluorescence imaging and histological analysis. Tumor cells invaded bone marrow spaces in the humanized bone and formed osteolytic lesions; osteoclastic bone resorption was, however, not reduced by ß1 integrin knockdown. Taken together, we demonstrate that ß1 integrins have a pivotal role in bone colonization using unique tissue-engineered humanized bone models.
Asunto(s)
Neoplasias Óseas/patología , Neoplasias de la Mama/patología , Integrina beta1/metabolismo , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/patología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ingeniería de TejidosRESUMEN
Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches.
Asunto(s)
Órganos Bioartificiales , Desarrollo Óseo/fisiología , Hematopoyesis/fisiología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Andamios del Tejido , Animales , Sustitutos de Huesos/síntesis química , Diferenciación Celular/fisiología , Células Cultivadas , Diseño de Equipo , Análisis de Falla de Equipo , Femenino , Humanos , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Osteoblastos/fisiología , Osteogénesis/fisiología , Nicho de Células Madre/fisiología , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodosRESUMEN
In vivo osteochondral defect models predominantly consist of small animals, such as rabbits. Although they have an advantage of low cost and manageability, their joints are smaller and more easily healed compared with larger animals or humans. We hypothesized that osteochondral cores from large animals can be implanted subcutaneously in rats to create an ectopic osteochondral defect model for routine and high-throughput screening of multiphasic scaffold designs and/or tissue-engineered constructs (TECs). Bovine osteochondral plugs with 4 mm diameter osteochondral defect were fitted with novel multiphasic osteochondral grafts composed of chondrocyte-seeded alginate gels and osteoblast-seeded polycaprolactone scaffolds, prior to being implanted in rats subcutaneously with bone morphogenic protein-7. After 12 weeks of in vivo implantation, histological and micro-computed tomography analyses demonstrated that TECs are susceptible to mineralization. Additionally, there was limited bone formation in the scaffold. These results suggest that the current model requires optimization to facilitate robust bone regeneration and vascular infiltration into the defect site. Taken together, this study provides a proof-of-concept for a high-throughput osteochondral defect model. With further optimization, the presented hybrid in vivo model may address the growing need for a cost-effective way to screen osteochondral repair strategies before moving to large animal preclinical trials.
Asunto(s)
Enfermedades Óseas , Huesos , Enfermedades de los Cartílagos , Condrocitos , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Enfermedades Óseas/patología , Enfermedades Óseas/terapia , Huesos/lesiones , Huesos/metabolismo , Huesos/patología , Cartílago/lesiones , Cartílago/metabolismo , Cartílago/patología , Enfermedades de los Cartílagos/patología , Enfermedades de los Cartílagos/terapia , Bovinos , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/patología , Condrocitos/trasplante , Modelos Animales de Enfermedad , Humanos , Poliésteres/química , Poliésteres/farmacología , Conejos , RatasRESUMEN
This study reports on an original concept of additive manufacturing for the fabrication of tissue engineered constructs (TEC), offering the possibility of concomitantly manufacturing a customized scaffold and a bioreactor chamber to any size and shape. As a proof of concept towards the development of anatomically relevant TECs, this concept was utilized for the design and fabrication of a highly porous sheep tibia scaffold around which a bioreactor chamber of similar shape was simultaneously built. The morphology of the bioreactor/scaffold device was investigated by micro-computed tomography and scanning electron microscopy confirming the porous architecture of the sheep tibiae as opposed to the non-porous nature of the bioreactor chamber. Additionally, this study demonstrates that both the shape, as well as the inner architecture of the device can significantly impact the perfusion of fluid within the scaffold architecture. Indeed, fluid flow modelling revealed that this was of significant importance for controlling the nutrition flow pattern within the scaffold and the bioreactor chamber, avoiding the formation of stagnant flow regions detrimental for in vitro tissue development. The bioreactor/scaffold device was dynamically seeded with human primary osteoblasts and cultured under bi-directional perfusion for two and six weeks. Primary human osteoblasts were observed homogenously distributed throughout the scaffold, and were viable for the six week culture period. This work demonstrates a novel application for additive manufacturing in the development of scaffolds and bioreactors. Given the intrinsic flexibility of the additive manufacturing technology platform developed, more complex culture systems can be fabricated which would contribute to the advances in customized and patient-specific tissue engineering strategies for a wide range of applications.