RESUMO
Albumin-based hydrogels are increasingly attractive in tissue engineering because they provide a xeno-free, biocompatible and potentially patient-specific platform for tissue engineering and drug delivery. The majority of research on albumin hydrogels has focused on bovine serum albumin (BSA), leaving human serum albumin (HSA) comparatively understudied. Different gelation methods are usually employed for HSA and BSA, and variations in the amino acid sequences of HSA and BSA exist; these account for differences in the hydrogel properties. Heat-induced gelation of aqueous HSA is the easiest method of synthesizing HSA hydrogels however hydrogel opacity and poor cell attachment limit their usefulness in downstream applications. Here, a solution to this problem is presented. Stable and translucent HSA hydrogels were created by controlled thermal gelation and the addition of sodium chloride. The resulting bio-inert hydrogel was then subjected to air plasma treatment which functionalised its surface, enabling the attachment of basement membrane matrix (Geltrex). In vitro survival and proliferation studies of foetal human osteoblasts subsequently demonstrated good biocompatibility of functionalised albumin hydrogels compared to untreated samples. Thus, air plasma treatment enables functionalisation of inert heat-derived HSA hydrogels with extracellular matrix proteins and these may be used as a xeno-free platform for biomedical research or cell therapy.
Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Gases em Plasma , Albumina Sérica Humana/química , Engenharia Tecidual/métodos , Materiais Biocompatíveis/toxicidade , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/toxicidade , Proteínas da Matriz Extracelular/ultraestrutura , Temperatura Alta , Humanos , Hidrogéis/toxicidade , Teste de Materiais , Microscopia Eletrônica de Varredura , Osteoblastos , Albumina Sérica Humana/toxicidade , Albumina Sérica Humana/ultraestrutura , Cloreto de Sódio/química , Propriedades de SuperfícieRESUMO
Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-ß induced protein (TGFßIp). The 4(th)_FAS1 domain of TGFßIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4(th)_FAS1 domains of TGFßIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αß conformation to ß-sheet oligomers. The ß-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that ß-oligomers of H572R were larger compared to R555W. The ß-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The ß-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.
Assuntos
Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/toxicidade , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/toxicidade , Fibroblastos/efeitos dos fármacos , Mutação , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/toxicidade , Sequência de Aminoácidos , Proteínas Amiloidogênicas/genética , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Substância Própria/citologia , Substância Própria/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas da Matriz Extracelular/genética , Fibroblastos/citologia , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Cultura Primária de Células , Desnaturação Proteica , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Fator de Crescimento Transformador beta/genéticaRESUMO
INTRODUCTION: The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA. METHODS: The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry. RESULTS: Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining. CONCLUSIONS: The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by HA injection was abrogated by Cd44 ablation, suggesting that interaction of the injected HA with CD44 is central to its protective effects on joint tissue remodeling and degeneration in OA progression.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/administração & dosagem , Osteoartrite/patologia , Viscossuplementos/administração & dosagem , Proteínas ADAM/biossíntese , Proteína ADAMTS5 , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/toxicidade , Fibrose , Imuno-Histoquímica , Injeções Intra-Arteriais , Masculino , Metaloproteinase 13 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Osteoartrite/metabolismo , Reação em Cadeia da Polimerase , Membrana Sinovial/irrigação sanguínea , Membrana Sinovial/patologia , Transcriptoma , Fator de Crescimento Transformador beta/toxicidadeRESUMO
Spatial patterning of proteins is a valuable technique for many biological applications and is the prevailing tool for defining microenvironments for cells in culture, a required procedure in developmental biology and tissue engineering research. However, it is still challenging to achieve protein patterns that closely mimic native microenvironments, such as gradient protein distributions with desirable mechanical properties. By combining projection dynamic mask lithography and protein engineering with non-canonical photosensitive amino acids, we demonstrate a simple, scalable strategy to fabricate any user-defined 2D or 3D stable gradient pattern with complex geometries from an artificial extracellular matrix (aECM) protein. We show that the elastic modulus and chemical nature of the gradient profile are biocompatible and allow useful applications in cell biological research.
Assuntos
Técnicas de Cultura de Células/métodos , Proteínas da Matriz Extracelular/química , Impressão/métodos , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Módulo de Elasticidade , Escherichia coli/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/toxicidade , Humanos , Teste de Materiais , Dados de Sequência Molecular , Células PC12 , RatosRESUMO
Relapsing polychondritis is an autoimmune disease that affects cartilage in the ear, nose, and respiratory tract. A pathogenic immune response has been proposed and antibodies to several cartilage proteins are detected in sera from these patients. To investigate the role of the humoral immune response in relapsing polychondritis, we used the matrilin-1-induced relapsing polychondritis model. Mice deficient of B cells (muMT) and mice congenic at the complement factor 5, were immunized with matrilin-1, a cartilage-specific protein mainly detected in the tracheal cartilage. To investigate the binding properties and tissue selection of matrilin-1-specific antibodies we produced matrilin-1-specific B-cell hybridomas. Although 83% of the micro MT heterozygous mice developed respiratory distress and erosive chondritis in the respiratory tract, none of the B-cell-deficient mice were susceptible to disease. In addition, we show that complement factor 5 is important for the induction of matrilin-1-induced relapsing polychondritis. Monoclonal matrilin-1-specific antibodies injected into neonatal mice bound specifically to cartilage of the respiratory tract and adult B-cell-deficient mice injected with the same antibodies developed erosive chondritis in the respiratory tract. We conclude that relapsing polychondritis can be mediated by a pathway involving tissue-specific antibodies and complement activation.
Assuntos
Anticorpos , Linfócitos B/imunologia , Complemento C5/imunologia , Proteínas da Matriz Extracelular/imunologia , Glicoproteínas/imunologia , Policondrite Recidivante/imunologia , Animais , Animais Recém-Nascidos , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Cartilagem/imunologia , Cartilagem/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Proteínas da Matriz Extracelular/toxicidade , Glicoproteínas/toxicidade , Hibridomas , Proteínas Matrilinas , Camundongos , Camundongos Congênicos , Policondrite Recidivante/induzido quimicamente , Sistema Respiratório/imunologiaRESUMO
PURPOSE: Vascular endothelial growth factor (VEGF) is a potent angiogenic agent and plays a major role in tumor growth and metastases. We have previously reported the locoregional (i.p.) delivery of adenovirus-mediated antiangiogenic soluble FLT-1 (sFLT-1; a naturally encoded potent VEGF antagonist) gene therapy to inhibit VEGF action in a murine ovarian carcinoma model. This study was predicated on the fact that systemic delivery of sFLT-1 might allow an approach for therapy of disseminated tumor. The purpose of this study is to test the effects of i.v. delivered, adenovirus-mediated sFLT-1 on the survival duration in a murine ovarian tumor model and to evaluate the safety of i.v.-delivered versus i.p.-delivered adenovirus-mediated sFLT-1 in non-tumor-bearing mice. EXPERIMENTAL DESIGN: To determine the effects of i.v.-administered adenovirus-mediated sFLT-1 on survival duration of mice bearing i.p. human ovarian tumors, an E1A/B-deleted, (replication-deficient) infectivity-enhanced recombinant adenovirus AdRGDGFPsFLT-1 encoding cDNA for both sFLT-1 and GFP (green fluorescent protein), a control adenovirus AdRGDGFP encoding GFP alone, or PBS was delivered i.v. The therapeutic effect of sFLT-1 was evaluated by survival duration of the mice. Furthermore, the safety of i.v.- or i.p.-delivered adenovirus-mediated sFLT-1 was evaluated by administering AdRGDGFPsFLT-1, AdRGDGFP, or PBS either i.v. or i.p. into non-tumor-bearing mice. Adenovirus-mediated gene expression was determined by determining GFP expression using fluorescent microscopy and by assessing sFLT-1 expression in liver, lungs, spleen, and kidneys by immunohistochemistry using anti-FLT-1 monoclonal antibody. Systemic levels of sFLT-1 were evaluated by ELISA and the toxicity was evaluated by histopathology. RESULTS: The i.v. delivery of AdRGDGFPsFLT-1 in the ovarian tumor model resulted in a shorter duration of survival of the mice as compared with the control group. Furthermore, in the safety evaluation experiment, i.v. administration of AdRGDGFPsFLT-1 in non-tumor-bearing mice principally localized to the liver. This localization lead to sFLT-1 overexpression, mainly in the liver, resulting in hemorrhage and tissue toxicity. However, i.p. delivery of AdRGDGFPsFLT-1 did not localize principally to the liver, leading to negligible expression of sFLT-1, and no intrahepatic hemorrhage or toxicity was observed. The i.v. delivery of the control virus AdRGDGFP also principally localized to the liver, leading to GFP expression mainly in the liver. However, neither hemorrhage nor morphological cytotoxicity was observed. i.p. delivery of AdRGDGFP resulted in ectopic localization to the liver with very little GFP expression and no toxicity. These results suggest that overexpression of sFLT-1 in the liver as a result of i.v. delivery is hepatotoxic. CONCLUSIONS: Our results suggest that i.v. delivery of the sFLT-1 gene via replication-deficient, infectivity-enhanced recombinant adenoviral vectors will result in overexpression of sFLT-1 in the liver leading to unacceptable hepatotoxicity. Tumor-specific targeting of the vectors and tumor-specific expression strategies should be used to ensure a clinically useful antiangiogenesis gene therapy.