RESUMO
Recently, the rapid development and application of mass spectrometry (MS)-based technologies have markedly improved the comprehensive proteomic characterization of global proteome and protein post-translational modifications (PTMs). However, the current conventional approach for global proteomic analysis is often carried out separately from PTM analysis. In our study, we developed an integrated workflow for multiplex analysis of global, glyco-, and phospho-proteomics using breast cancer patient-derived xenograft (PDX) tumor samples. Our approach included the following steps: trypsin-digested tumor samples were enriched for phosphopeptides through immobilized metal ion affinity chromatography (IMAC), followed by enrichment of glycopeptides through mixed anion exchange (MAX) method, and then the flow-through peptides were analyzed for global proteomics. Our workflow demonstrated an increased identification of peptides and associated proteins in global proteome, as compared to those using the peptides without PTM depletion. In addition to global proteome, the workflow identified phosphopeptides and glycopeptides from the PTM enrichment. We also found a subset of glycans with unique distribution profiles in the IMAC flow-through, as compared to those enriched directly using the MAX method. Our integrated workflow provided an effective platform for simultaneous global proteomic and PTM analysis of biospecimens.
Assuntos
Neoplasias da Mama/química , Glicopeptídeos/análise , Fosfopeptídeos/análise , Proteoma/análise , Proteômica/métodos , Fluxo de Trabalho , Animais , Cromatografia Líquida , Xenoenxertos/química , Humanos , Camundongos , Proteólise , Proteoma/química , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
PURPOSE: The purpose of this study was to investigate the feasibility of in vivo imaging of human pancreatic ductal cells by OATP1B3 reporter gene under MRI. METHODS: A human cell line (PANC-1) derived from the pancreatic ductal epithelium was used in this study. After transduction of OATP1B3, the cellular physiological functions and the ability of intracellular uptake of the MRI contrast medium (Gd-EOB-DTPA) were examined. Induced differentiation of the PANC-1 cells into hormone-secreting cells were performed to simulate pancreatic ß-like cells. The hormone-secreting cells were implanted into rats and in vivo MRI was evaluated. RESULTS: The mRNA and proteins of OATP1B3 were highly expressed. No significant change of cellular physiological functions was found after the expression. After induced differentiation, the hormone secretion capacities of the OATP1B3-expressing PANC-1 cells were confirmed. Intra-cellular uptake of Gd-EOB-DTPA was determined in vitro by inductively coupled plasma mass spectrometry and MRI. In vivo MRI of the OATP1B3-expressing xenograft revealed an increased signal intensity after contrast enhancement. CONCLUSION: OATP1B3 can be used as a safe and feasible in vivo MRI gene reporter for human pancreatic ductal cells.
Assuntos
Genes Reporter/genética , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Imageamento por Ressonância Magnética/métodos , Animais , Linhagem Celular , Meios de Contraste , Estudos de Viabilidade , Feminino , Gadolínio DTPA , Xenoenxertos/química , Xenoenxertos/diagnóstico por imagem , Xenoenxertos/metabolismo , Humanos , Células Secretoras de Insulina/química , Camundongos , Camundongos SCID , Imagem Molecular , Ratos , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/química , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismoRESUMO
Bone grafting is the second most common tissue transplantation procedure worldwide. One of the alternative methods for bone repair under investigation is a tissue-engineered bone substitute. An ideal property of tissue-engineered bone substitutes is osteoinductivity, defined as the ability to stimulate primitive cells to differentiate into a bone-forming lineage. In the current study, we use a decellularization and oxidation protocol to produce a porcine bone scaffold and examine whether it possesses osteoinductive potential and can be used to create a tissue-engineered bone microenvironment. The decellularization protocol was patented by our lab and consists of chemical decellularization and oxidation steps using combinations of deionized water, trypsin, antimicrobials, peracetic acid, and triton-X100. To test if the bone scaffold was a viable host, preosteoblasts were seeded and analyzed for markers of osteogenic differentiation. The osteoinductive potential was observed in vitro with similar osteogenic markers being expressed in preosteoblasts seeded on the scaffolds and demineralized bone matrix. To assess these properties in vivo, scaffolds with and without preosteoblasts preseeded were subcutaneously implanted in mice for 4 weeks. MicroCT scanning revealed 1.6-fold increased bone volume to total volume ratio and 1.4-fold increase in trabecular thickness in scaffolds after implantation. The histological analysis demonstrates new bone formation and blood vessel formation with pentachrome staining demonstrating osteogenesis and angiogenesis, respectively, within the scaffold. Furthermore, CD31+ staining confirmed the endothelial lining of the blood vessels. These results demonstrate that porcine bone maintains its osteoinductive properties after the application of a patented decellularization and oxidation protocol developed in our laboratory. Future work must be performed to definitively prove osteogenesis of human mesenchymal stem cells, biocompatibility in large animal models, and osteoinduction/osseointegration in a relevant clinical model in vivo. The ability to create a functional bone microenvironment using decellularized xenografts will impact regenerative medicine, orthopedic reconstruction, and could be used in the research of multiple diseases.
Assuntos
Xenoenxertos/transplante , Células-Tronco Mesenquimais/metabolismo , Alicerces Teciduais/química , Transplante Heterólogo , Animais , Substitutos Ósseos/química , Diferenciação Celular , Linhagem Celular , Xenoenxertos/química , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Osteoblastos , Osteogênese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Suínos , Engenharia Tecidual/métodosRESUMO
Janus polymerization is a novel and efficient way to synthesize diblock and multiblock copolymers in one step by using Lu(OTf)3 and propylene epoxide as a catalytic system. By modifying the epoxide initiator, which is located at the block junction with various functional groups, the possibility for future topological design is provided. Herein, 2-bicyclo[2.2.1]hept-5-en-2-yl oxirane (NB-EO) is used as an alternative for propylene epoxide to synthesize a poly(THF-co-CL)-b-PCL diblock copolymer featuring a norbornene group at this position. This also results in multiblock [poly(THF-co-CL)-b-PCL]m copolymers carrying multiple norbornene moieties through Janus polymerization. Subsequent ring-opening metathesis copolymerization (ROMP) of the resulting poly(THF-co-CL)-b-PCL macromonomers with norbornenyl-terminated polysarcosine (PSar-NB) allows the facile preparation of heterograft molecular polymer brushes (MPBs). The MPBs feature three heterografts of PCL, P(CL-co-THF) and PSar, potentially equipping these structures with biocompatibility, biodegradability, semi-crystallinity, and amphiphilicity. A phase separation is observed after annealing in both TEM and AFM analysis. Due to their amphiphilic nature, the MPBs also undergo self-assembly into micelles in aqueous solution. Such materials combining polypeptoids, polyesters, and polyethers segments are expected to attract wide attention in drug delivery applications.
Assuntos
Butileno Glicóis/química , Xenoenxertos/química , Peptídeos/química , Poliésteres/química , Polímeros/química , Estrutura Molecular , PolimerizaçãoRESUMO
BACKGROUND: Glutaraldehyde-treated pericardia for cardiovascular applications have poor long-term clinical results. The efficacy of a combined physical/chemical treatment to improve pericardium biocompatibility and vascular regeneration was assessed and compared with detergent treatment and two commercial bovine pericardia: PeriGuard (DGBP) and Edwards pericardium (nDGBP). The physical and chemical process was applied to bovine and human pericardia (DBP-DHP), and the detergent process was applied to bovine (DDBP). MATERIAL AND METHODS: Native (NBP) and treated bovine tissues were assessed for decellularization (HE/DAPI/DNA/α-Gal and MHC-1 staining) and mechanical integrity ex vivo. Twenty Wistar rats received subcutaneous patches of each bovine tissue to assess immunogenic response up to 4 months (flow cytometry). Ten additional rats received four subcutaneous bovine-treated patches (one/condition) to evaluate the inflammatory reaction (CD3/CD68 immunostaining), calcification (von Kossa staining/calcium quantification), and integration assessment (Hematoxylin and eosin staining). Finally, 15 rodents received a patch on the aorta (DBP n = 5, DHP n = 5, and DGBP n = 5), and vascular biocompatibility and arterial wall regeneration were assessed after 4 months (CD3/CD68/CD31/ASMA and Miller staining). RESULTS: DBP reached the higher level of decellularization, no immunogenic response whereas maintaining mechanical properties. DBP induced the lowest level grade of inflammation after 2 months (P < 0.05) concomitantly for better remodeling. No complications occurred with DBP and DHP where vascular regeneration was confirmed. Moreover, they induced a low level of CD3/CD68 infiltrations. CONCLUSIONS: This process significantly reduces immunogenicity and improves biocompatibility of bovine and human pericardia for better vascular regeneration.
Assuntos
Aorta/fisiologia , Aorta/cirurgia , Pericárdio/transplante , Regeneração/imunologia , Animais , Bovinos , DNA/análise , Feminino , Xenoenxertos/química , Humanos , Masculino , Teste de Materiais , Pericárdio/imunologia , Ratos WistarRESUMO
OBJECTIVE: Uterine sarcomas (US) and carcinosarcomas (CS) are rare, aggressive cancers. The lack of reliable preclinical models hampers the search for new treatment strategies and predictive biomarkers. To this end, we established and characterized US and CS patient-derived xenograft (PDX) models. METHODS: Tumor fragments of US and CS were subcutaneously implanted into immunocompromised mice. Engrafted xenograft and original tumors were compared by means of histology, immunohistochemistry, whole-genome low-coverage sequencing for copy number variations, and RNA sequencing. RESULTS: Of 13 implanted leiomyosarcomas (LMS), 10 engrafted (engraftment rate of 77%). Also 2 out of 7 CS (29%) and one high-grade US (not otherwise specified) models were successfully established. LMS xenografts showed high histological similarity to their corresponding human tumors. Expression of desmin and/or H-caldesmon was detected in 8/10 LMS PDX models. We noticed that in CS models, characterized by the concomitant presence of a mesenchymal and an epithelial component, the relative distribution of the components is varying over the generations, as confirmed by changes in vimentin and cytokeratin expression. The similarity in copy number profiles between original and xenograft tumors ranged from 57.7% to 98.2% for LMS models and from 47.4 to 65.8% for CS models. Expression pattern stability was assessed by clustering RNA expression levels of original and xenograft tumors. Six xenografts clustered together with their original tumor, while 3 (all LMS) clustered apart. CONCLUSIONS: We present here a panel of clinically annotated uterine sarcoma and carcinosarcoma PDX models, which will be a useful tool for preclinical testing of new therapies.
Assuntos
Carcinossarcoma/patologia , DNA de Neoplasias/análise , Modelos Animais de Doenças , Xenoenxertos/patologia , Leiomiossarcoma/patologia , RNA Neoplásico/análise , Neoplasias Uterinas/patologia , Adulto , Idoso , Animais , Proteínas de Ligação a Calmodulina/análise , Carcinossarcoma/química , Carcinossarcoma/genética , Variações do Número de Cópias de DNA , Desmina/análise , Feminino , Expressão Gênica , Sobrevivência de Enxerto , Xenoenxertos/química , Humanos , Leiomiossarcoma/química , Leiomiossarcoma/genética , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Análise de Sequência de RNA , Transplante Heterólogo , Neoplasias Uterinas/química , Neoplasias Uterinas/genéticaRESUMO
OBJECTIVE: To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2), and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo. METHODS: The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study. Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM), respectively. The supernatants were collected and used to culture hASCs in vitro. Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 14 and 21 days, the calcification deposition was determined by alizarin red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study, 6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups: (1) BioCaP scaffold only, (2) BioCaP scaffold+hASCs, (3) BMP-2-BioCaP scaffold, (4) BMP-2-BioCaP scaffold+hASCs (test group). After 4 weeks of implantation, hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight, plate-like and sharp-edged crystal units, and the length of the crystal units varied between 5 and 10 µm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days, and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2-BioCaP. ALP activity was higher by the induction of OM+BMP-2-BioCaP than of the other groups (P<0.01). More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (OPN) and osteocalcin (OC) were determined in the OM+BMP-2-BioCaP group at different time points (P<0.01). HE staining showed that, in the test group and BMP-2-BioCaP scaffold group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the BMP-2-BioCaP scaffold group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups. No obvious positive results were found in the other groups. CONCLUSION: BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo. The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 2/farmacocinética , Calcificação Fisiológica/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Fosfatos de Cálcio/farmacocinética , Liberação Controlada de Fármacos/efeitos dos fármacos , Transplante de Células-Tronco Mesenquimais/métodos , Osteogênese/efeitos dos fármacos , Engenharia Tecidual/métodos , Tecido Adiposo , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Proteína Morfogenética Óssea 2/administração & dosagem , Proteína Morfogenética Óssea 2/uso terapêutico , Osso e Ossos , Fosfatos de Cálcio/administração & dosagem , Fosfatos de Cálcio/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Xenoenxertos/química , Xenoenxertos/fisiologia , Xenoenxertos/transplante , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Osteopontina/efeitos dos fármacos , Osteopontina/metabolismo , Propriedades de Superfície , Alicerces Teciduais/químicaRESUMO
The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC-MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation (source code is available from http://homepages.uc.edu/~wang2x7/Research.htm ). From these assessments, we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61 to 93% of the time. When comparing across different instruments and quantitative technologies, using multiple replicates, differential genes were reproduced by other data sets from 67 to 99% of the time. Projecting gene differences to biological pathways and networks increased the degree of similarity. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation.
Assuntos
Xenoenxertos/química , Proteômica/métodos , Proteômica/normas , Neoplasias da Mama/química , Neoplasias da Mama/metabolismo , Cromatografia Líquida , Interpretação Estatística de Dados , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Redes e Vias Metabólicas , Variações Dependentes do Observador , Proteoma , Proteômica/instrumentação , Controle de Qualidade , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/normasRESUMO
Few targeted drugs were approved for treatment of colorectal cancer (CRC). Cyclin-dependent kinase 8 played a vital role in regulating transcription and was a key colorectal oncogene associated to colorectal cancer. Here, through de novo drug design and in depth structure-activity relationship analysis, title compound 22, (3-(3-(1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)-N-(4-methyl-3-(trifluoromethyl)phenyl)propenamide), was discovered as a potent type II CDK8 inhibitor, which exhibited potent kinase activity with an IC50 value of 48.6 nM and could significantly inhibit tumor growth in xenografts of CRC in vivo. Further mechanism studies indicated that it could target CDK8 to indirectly inhibit ß-catenin activity, which caused downregulation of the WNT/ß-catenin signal and inducing cell cycle arrest in G2/M and S phases. More importantly, the title compound exhibited low toxicity with good bioavailability (F = 39.8%). These results could provide the reference for design of new type II CDK8 inhibitors against colorectal cancer.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/patologia , Quinase 8 Dependente de Ciclina , Desenho de Fármacos , Xenoenxertos/química , Xenoenxertos/metabolismo , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Relação Estrutura-Atividade , beta Catenina/metabolismoRESUMO
The mouse transplantation model remains the most relevant methodology to assess the functional capacities of mammary cells and is particularly appropriate for investigations regarding mammary stem cells, whatever the species studied. Following xenotransplantation in mice mammary fat pad, the development of the xenograft is commonly evaluated by immunohistology. Here, we present a simple and rapid method to control the species specificity of a xenograft based on genomic DNA PCR amplification. DNA is extracted from the fixed samples intended for histology, thus allowing the reuse of precious samples. Standard and digital droplet PCR (requiring low DNA quantities) methods have been used to make the present method suitable for the analysis of xenotransplanted samples.
Assuntos
Genômica/métodos , Xenoenxertos , Glândulas Mamárias Animais , Reação em Cadeia da Polimerase/métodos , Animais , Bovinos , DNA/análise , DNA/genética , DNA/metabolismo , Feminino , Xenoenxertos/química , Xenoenxertos/crescimento & desenvolvimento , Xenoenxertos/metabolismo , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Camundongos , Transplante HeterólogoRESUMO
SUMMARY: Tissue engineering aims to fabricate a scaffold that exhibits a suitable surface topography for a desired cellular response. Therefore, a study analyzing the characteristics of bone grafts is important for future research directions. This work aims to analyze the physical-chemical characteristics of commercially available bone grafts of human and bovine origin for dental use, using morphological analysis of the surface and chemical composition by variable pressure scanning electron microscope (VP-SEM) and energy-dispersive x-ray (EDX) spectrometry. In addition, pore diameter and surface area were analyzed by degassing method using a porosimeter, and particle size by laser diffraction. The analyzed allograft and xenograft particles differ in morphological characteristics and chemical composition. The allograft particles present a cuboidal and prismatic geometric morphology with angled edges and the absence of macropores. On the contrary, the xenograft particles present an irregular morphology with macropores in their structure. There is a statistically significant difference in C, P, and Ca between the xenograft and allografts (p < 0,05). The analyzed composition of allografts showed mainly the presence of C and O. In contrast, the composition of the xenograft was mainly Ca. These differences could influence the osteogenic properties of allografts and xenografts. This analysis provides basic information to understand the physicochemical properties of allografts and xenografts that facilitate cell-graft interaction.
La ingeniería de tejidos tiene como objetivo fabricar un andamio que muestre una topografía de superficie adecuada para una respuesta celular deseada. Por tanto, un estudio que analice las características de los injertos óseos es importante para futuros enfoques de investigación. Este trabajo tiene como objetivo analizar las características físico-químicas de injertos óseos de origen humano y bovino disponibles comercialmente para uso odontológico, mediante análisis morfológico de la superficie y composición química mediante microscopio electrónico de barrido de presión variable (VP-SEM) y x-dispersivo de energía. espectrometría de rayos (EDX). Además, el diámetro de los poros y el área superficial se analizaron mediante el método de desgasificación utilizando un porosímetro y el tamaño de las partículas mediante difracción láser. Las partículas de aloinjerto y xenoinjerto analizadas difieren en características morfológicas y composición química. Las partículas del aloinjerto presentan una morfología geométrica cúbica y prismática con bordes angulados y ausencia de macroporos. Por el contrario, las partículas de xenoinjerto presentan una morfología irregular con macroporos en su estructura. Existe una diferencia estadísticamente significativa en C, P y Ca entre el xenoinjerto y los aloinjertos (p < 0,05). La composición analizada de los aloinjertos mostró principalmente la presencia de C y O. Por el contrario, la composición del xenoinjerto fue principalmente Ca. Estas diferencias podrían influir en las propiedades osteogénicas de los aloinjertos y xenoinjertos. Este análisis proporciona información básica para comprender las propiedades fisicoquímicas de aloinjertos y xenoinjertos que facilitan la interacción célula-injerto.
Assuntos
Humanos , Animais , Bovinos , Aloenxertos/anatomia & histologia , Aloenxertos/química , Espectrometria por Raios X , Regeneração Óssea , Microscopia Eletrônica de Varredura , Porosidade , Transplante Ósseo , Xenoenxertos/anatomia & histologia , Xenoenxertos/químicaRESUMO
Numerous animal species have been proposed as sources of corneal tissue for obtaining decellularized xenografts. The selection of an appropriate animal model must take into consideration the differences in the composition and structure of corneal proteins between humans and other animal species in order to minimize immune response and improve outcome of the xenotransplant. Here, we compared the amino-acid sequences of 16 proteins present in the corneal stromal matrix of 14 different animal species using Basic Local Alignment Search Tool, and calculated a similarity score compared to the respective human sequence. Primary amino acid structures, isoelectric point and grand average of hydropathy (GRAVY) values of the 7 most abundant proteins (i.e. collagen α-1 (I), α-1 (VI), α-2 (I) and α-3 (VI), as well as decorin, lumican, and keratocan) were also extracted and compared to those of human. The pig had the highest similarity score (91.8%). All species showed a lower proline content compared to human. Isoelectric point of pig (7.1) was the closest to the human. Most species have higher GRAVY values compared to human except horse. Our results suggest that porcine cornea has a higher relative suitability for corneal transplantation into humans compared to other studied species.
Assuntos
Transplante de Córnea , Xenoenxertos/química , Transplante Heterólogo , Algoritmos , Animais , Colágeno/química , Biologia Computacional , Decorina/química , Matriz Extracelular/química , Proteínas do Olho/química , Cavalos , Humanos , Ponto Isoelétrico , Lumicana/química , Transplante de Neoplasias , Filogenia , Prolina/química , Proteoglicanas/química , Alinhamento de Sequência , Especificidade da Espécie , SuínosRESUMO
BACKGROUND: The further functionalization of natural existing biomaterials is a very efficient method to introduce additional advanced characteristics on a unique structural composition and architecture. OBJECTIVE: As an example, different animal sources, if properly treated, can be used to develop bone xenograft active in hard tissues regeneration. In this sense, it is also important to consider that the selected process has to take into consideration the intrinsic variability of the base material itself and possibly being able to compensate for it. METHODS: In this work we characterize cancellous bovine bone treated by deposition of polymer and collagen and we show that the added components not only lead to a more resistant and more hydrophilic material, but also reduce the conventional correlation between apparent density and elastic modulus, which, in general, is a major source of uncertainty and risk in xenografts usage. RESULTS: Moreover, though intrinsically reinforcing the material, the deposition process leaves the specific open-porous structure, that allows cells proliferation and vessels ingrowth, basically unaltered. CONCLUSION: The final material combines in a single piece and at the same time, mechanical resistance, homogeneous mechanical response and proper structural characteristics that allow further integration within the patient autochthonous tissues.
Assuntos
Regeneração Óssea , Substitutos Ósseos/química , Osso e Ossos/química , Xenoenxertos/química , Engenharia Tecidual/métodos , Animais , Substitutos Ósseos/metabolismo , Osso e Ossos/metabolismo , Bovinos , Proliferação de Células , Colágeno/química , Colágeno/metabolismo , Módulo de Elasticidade , Xenoenxertos/metabolismo , Teste de Materiais , PorosidadeRESUMO
BACKGROUND: Persistent organic pollutants (POPs) are known to accumulate in adipose tissues (AT). This storage may be beneficial by diverting POPs from other sensitive tissues or detrimental because of chronic release of pollutants as indirectly suggested during weight loss. The aim is to study the biological and/or toxic effects that chronic POP release from previously contaminated grafted AT could exert in a naïve mouse. METHODS: C57BL/6J male mice were exposed intraperitoneally to 2,3,7,8-tetrachlorodibenzo-p-doxin (TCDD); their epididymal fat pads were collected and grafted on the back skin of uncontaminated recipient mice whose brain, liver, and epididymal ATs were analyzed (TCDD concentration, relevant gene expression). Kinetics of release and redistribution were modeled using Physiologically Based PharmacoKinetics (PBPK). RESULTS: The grafts released TCDD over a period of 10â¯weeks with different kinetics of distribution in the three organs studied. A PBPK model was used to simulate the AT releasing process and the incorporation of TCDD into the major organs. At three weeks post-graft, we observed significant changes in gene expression in the liver and the host AT with signatures reminiscent of inflammation, gluconeogenesis and fibrosis as compared to the control. CONCLUSIONS: This study confirms that AT-stored TCDD can be released and distributed to the organs of the recipient hence leading to distinct changes in gene expression. This original model provides direct evidence of the potential toxic-relevant effects when endogenous sources of contamination are present.
Assuntos
Tecido Adiposo , Xenoenxertos , Dibenzodioxinas Policloradas , Tecido Adiposo/química , Tecido Adiposo/metabolismo , Tecido Adiposo/transplante , Animais , Encéfalo/metabolismo , Xenoenxertos/química , Xenoenxertos/metabolismo , Xenoenxertos/transplante , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dibenzodioxinas Policloradas/metabolismo , Dibenzodioxinas Policloradas/farmacocinética , Dibenzodioxinas Policloradas/toxicidadeRESUMO
Ultrasound molecular imaging (USMI) is accomplished by detecting microbubble (MB) contrast agents that have bound to specific biomarkers, and can be used for a variety of imaging applications, such as the early detection of cancer. USMI has been widely utilized in preclinical imaging in mice; however, USMI in humans can be challenging because of the low concentration of bound MBs and the signal degradation caused by the presence of heterogenous soft tissue between the transducer and the lesion. Short-lag spatial coherence (SLSC) beamforming has been proposed as a robust technique that is less affected by poor signal quality than standard delay-and-sum (DAS) beamforming. In this paper, USMI performance was assessed using contrast-enhanced ultrasound imaging combined with DAS (conventional CEUS) and with SLSC (SLSC-CEUS). Each method was characterized by flow channel phantom experiments. In a USMI-mimicking phantom, SLSC-CEUS was found to be more robust to high levels of additive thermal noise than DAS, with a 6dB SNR improvement when the thermal noise level was +6dB or higher. However, SLSC-CEUS was also found to be insensitive to increases in MB concentration, making it a poor choice for perfusion imaging. USMI performance was also measured in vivo using VEGFR2-targeted MBs in mice with subcutaneous human hepatocellular carcinoma tumors, with clinical imaging conditions mimicked using a porcine tissue layer between the tumor and the transducer. SLSC-CEUS improved the SNR in each of ten tumors by an average of 41%, corresponding to 3.0dB SNR. These results indicate that the SLSC beamformer is well-suited for USMI applications because of its high sensitivity and robust properties under challenging imaging conditions.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Modelos Biológicos , Imagem Molecular/métodos , Ultrassonografia/métodos , Animais , Artefatos , Xenoenxertos/química , Xenoenxertos/diagnóstico por imagem , Humanos , Camundongos , Neoplasias Experimentais/química , Neoplasias Experimentais/diagnóstico por imagem , Imagens de Fantasmas , Sensibilidade e Especificidade , Razão Sinal-Ruído , Suínos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análise , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Xenograft, namely bone-derived biological apatite (BAp), is widely recognized as a favorable biomaterial in bone tissue engineering owing to its biodegradability, biocompatibility, and osteoconductive properties. Substitutions of endogenous trace ions are thought to improve the osteogenic capacity of xenograft compared with synthetic hydroxyapatite (HAp). In order to modify the physicochemical and biological properties of apatite, different approaches to induce trace ion incorporation have been widely considered. In this study, we demonstrated that the incorporation of fluoride ions into porcine bone-derived biological apatite (pBAp) contributes to altered crystal morphology of the apatite, the sustained release of fluoride, and the in situ release of endogenous trace ions (e.g., magnesium and calcium) into the peripheral tissue microenvironment. This ionic balanced perimaterial microenvironment not only led to superior proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs), but also accelerated new bone formation of the calvarial defect on a rat model via the activation of Wnt/ß-catenin signaling. These promising observations may be attributed to the controlled release of endogenous trace ions from the xenograft to the peripheral tissue microenvironment driven by fluoride ion incorporation. Lastly, this study may provide a new insight to strengthen the osteogenicity of xenografts for clinical applications in the future.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Osso e Ossos/cirurgia , Cátions Bivalentes/metabolismo , Fluoretos/farmacologia , Xenoenxertos/química , Osteogênese/efeitos dos fármacos , Animais , Osso e Ossos/citologia , Osso e Ossos/fisiologia , Proliferação de Células/efeitos dos fármacos , Durapatita/química , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
OBJECTIVE: Docetaxel (DTX) remains the only effective drug for prolonging survival and improving quality of life of metastatic castration-resistant prostate cancer (mCRPC) patients. Combination anticancer therapy encapsulating DTX and another extract of traditional Chinese medicine is one nano-sized drug delivery system promising to generate synergistic anticancer effects, to maximize the treatment effect, and to overcome multi-drug resistance. The purpose of this study is to construct lipid-polymer hybrid nanoparticles (LPNs) as nanomedicine for co-encapsulation of DTX and curcumin (CUR). METHODS: DTX and CUR co-encapsulated LPNs (DTX-CUR-LPNs) were constructed. DTX-CUR-LPNs were evaluated in terms of particles size, zeta potential, drug encapsulation, and drug delivery. The cytotoxicity of the LPNs was evaluated on PC-3 human prostate carcinoma cells (PC3 cells) by MTT assays. In vivo anti-tumor effects were observed on the PC3 tumor xenografts in mice. RESULTS: The particle size of DTX-CUR-LPNs was 169.6 nm with a positive zeta potential of 35.7 mV. DTX-CUR-LPNs showed highest cytotoxicity and synergistic effect of two drugs in tumor cells in vitro. In mice-bearing PC-3 tumor xenografts, the DTX-CUR-LPNs inhibited tumor growth to a greater extent than other contrast groups, without inducing any obvious side effects. CONCLUSION: According to these results, the novel nanomedicine offers great promise for the dual drugs delivery to the prostate cancer cells, showing the potential of synergistic combination therapy for prostate cancer.
Assuntos
Curcumina/administração & dosagem , Xenoenxertos/efeitos dos fármacos , Lipídeos/química , Nanomedicina/métodos , Polímeros/química , Neoplasias da Próstata/tratamento farmacológico , Taxoides/administração & dosagem , Animais , Linhagem Celular Tumoral , Curcumina/farmacologia , Docetaxel , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Xenoenxertos/química , Humanos , Masculino , Camundongos , Tamanho da Partícula , Neoplasias da Próstata/química , Taxoides/farmacologiaRESUMO
The development of a reliable dose monitoring system in hadron therapy is essential in order to control the treatment plan delivery. Positron Emission Tomography (PET) is the only method used in clinics nowadays for quality assurance. However, the accuracy of this method is limited by the loss of signal due to the biological washout processes. Up to the moment, very few studies measured the washout processes and there is no database of washout data as a function of the tissue and radioisotope. One of the main difficulties is related to the complexity of such measurements, along with the limited time slots available in hadron therapy facilities. Thus, in this work, we proposed an alternative in vivo methodology for the measurement and modeling of the biological washout parameters without any radiative devices. It consists in the implementation of a point-like radioisotope source by direct injection on the tissues of interest and its measurement by means of high-resolution preclinical PET systems. In particular, the washout of 11C carbonate radioisotopes was assessed, considering that 11C is is the most abundant ß+ emitter produced by carbon beams. 11C washout measurements were performed in several tissues of interest (brain, muscle and 9L tumor xenograf) in rodents (Wistar rat). Results show that the methodology presented is sensitive to the washout variations depending on the selected tissue. Finally, a first qualitative correlation between 11C tumor washout properties and tumor metabolism (via 18F-FDG tracer uptake) was found.
Assuntos
Radioisótopos de Carbono/uso terapêutico , Fluordesoxiglucose F18/uso terapêutico , Neoplasias/diagnóstico por imagem , Perfusão/métodos , Animais , Encéfalo/efeitos dos fármacos , Radioisótopos de Carbono/química , Fluordesoxiglucose F18/química , Xenoenxertos/química , Humanos , Músculo Esquelético/química , Músculo Esquelético/efeitos dos fármacos , Neoplasias/química , Tomografia por Emissão de Pósitrons , Ratos , Distribuição TecidualRESUMO
The goal of the present work was to investigate the relationship between in vivo healing and inflammatory response and in vitro cytokine expression by macrophages of a synthetic bone filler (25% hydroxylapatite-75% ß-tricalcium phosphate) bearing a surface nanolayer of collagen. A clinically accepted, state-of-the-art xenograft material was used as a "negative control," that is, as a material that provides the correct clinical response for the intended use. In vitro data show that both materials exert a very low stimulation of proinflammatory cytokines by macrophages, and this was confirmed by the very mild inflammatory response detected in in vivo tests of local response in a rabbit model. Also, in vitro findings suggest a different mechanism of healing for the test and the control material, with a higher regenerative activity for the synthetic, resorbable filler, as confirmed by in vivo observation and literature reports. Thus, the simple in vitro model adopted provides a reasonable forecast of in vivo results, suggesting that new product development can be guided by in vitro tuning of cell-materials interactions.
Assuntos
Substitutos Ósseos/química , Fosfatos de Cálcio/química , Colágeno/química , Citocinas/metabolismo , Durapatita/química , Animais , Materiais Biomiméticos , Regeneração Óssea/efeitos dos fármacos , Bovinos , Fraturas do Fêmur/terapia , Perfilação da Expressão Gênica , Xenoenxertos/química , Inflamação , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Teste de Materiais , Coelhos , Propriedades de Superfície , Resultado do TratamentoRESUMO
Human tumor xenografts in immunodeficient mice are a very popular model to study the development of cancer and to test new drug candidates. Among the parameters analyzed are the variations in the lipid composition, as they are good indicators of changes in the cellular metabolism. Here, we present a study on the distribution of lipids in xenografts of NCI-H1975 human lung cancer cells, using MALDI imaging mass spectrometry and UHPLC-ESI-QTOF. The identification of lipids directly from the tissue by MALDI was aided by the comparison with identification using ESI ionization in lipid extracts from the same xenografts. Lipids belonging to PCs, PIs, SMs, DAG, TAG, PS, PA, and PG classes were identified and their distribution over the xenograft was determined. Three areas were identified in the xenograft, corresponding to cells in different metabolic stages and to a layer of adipose tissue that covers the xenograft.