RESUMO
Biomimetic proteoglycans (BPGs) were designed to mimic the three-dimensional (3D) bottlebrush architecture of natural extracellular matrix (ECM) proteoglycans, such as aggrecan. BPGs were synthesized by grafting native chondroitin sulfate bristles onto a synthetic poly(acrylic acid) core to form BPGs at a molecular weight of approximately â¼1.6â¯MDa. The aggrecan mimics were characterized chemically, physically, and structurally, confirming the 3D bottlebrush architecture as well as a level of water uptake, which is greater than that of the natural proteoglycan, aggrecan. Aggrecan mimics were cytocompatible at physiological concentrations. Fluorescently labeled BPGs were injected into the nucleus pulposus of the intervertebral disc ex vivo and were retained in tissue before and after static loading and equilibrium conditioning. BPGs infiltrated the tissue, distributed and integrated with the ECM on a molecular scale, in the absence of a bolus, thus demonstrating a new molecular approach to tissue repair: molecular matrix engineering. Molecular matrix engineering may compliment or offer an acellular alternative to current regenerative medicine strategies. STATEMENT OF SIGNIFICANCE: Aggrecan is a natural biomolecule that is essential for connective tissue hydration and mechanics. Aggrecan is composed of negatively charged chondroitin sulfate bristles attached to a protein core in a bottlebrush configuration. With age and degeneration, enzymatic degradation of aggrecan outpaces cellular synthesis resulting in a loss of this important molecule. We demonstrate a novel biomimetic molecule composed of natural chondroitin sulfate bristles grafted onto an enzymatically-resistant synthetic core. Our molecule mimics a 3D architecture and charge density of the natural aggrecan, can be delivered via a simple injection and is retained in tissue after equilibrium conditioning and loading. This novel material can serve as a platform for molecular repair, drug delivery and tissue engineering in regenerative medicine approaches.
Assuntos
Resinas Acrílicas/química , Agrecanas/química , Materiais Biomiméticos/química , Sulfatos de Condroitina/química , Matriz Extracelular/química , Fibroblastos/metabolismo , Teste de Materiais , Animais , Linhagem Celular , Fibroblastos/citologia , CamundongosRESUMO
A new combined imaging and chemical detection sensor for the measurement of localized L-glutamate release at the insect neuromuscular junction (NMJ) is presented. The sensor is comprised of an L-glutamate-sensitive fluorescent gel, spin-coated onto the tip of an optical imaging fiber. The gel is composed of L-glutamate oxidase (GLOD); a pH-sensitive fluorescent dye, SNAFL; and poly(acrylamide-co-N-acryloxysuccinimide) (PAN). NH(3) is liberated from the interaction of L-glutamate with GLOD, which reversibly reduces the emitted fluorescence signal from SNAFL. This sensor has a spatial resolution of 3-4 micro m, and an L-glutamate detection limit of between 10 and 100 micro M. L-glutamate release and re-uptake from the foregut plexus of Manduca sexta was detected by the sensor in the presence of the L-glutamate re-uptake blocker dihydrokainate, and the post-synaptic L-glutamate receptor antagonist CNQX.
Assuntos
Técnicas Biossensoriais/métodos , Ácido Glutâmico/metabolismo , Manduca/metabolismo , Junção Neuromuscular/metabolismo , Animais , Técnicas Biossensoriais/instrumentação , Fluorescência , Ácido Glutâmico/análise , Manduca/química , Junção Neuromuscular/químicaRESUMO
Chondroitin sulfate (CS) based bottle brush proteoglycan mimetics may be employed to restore tissue functionality. Synthesis of CS bottle brush structures requires immobilization of CS at its terminal end. In this study, we investigated commercially available natural CS for use in CS bottle brush synthesis. A terminal primary amine on CS was identified and utilized to conjugate amine-reactive vinyl monomers (i.e. acrylic acid and allyl glycidyl ether). Conjugation of vinyl monomers to the CS terminal amine was confirmed using a fluorescamine assay, (1)H NMR, and ATR-FTIR. CS was also immobilized onto epoxy functionalized surfaces via the CS terminal primary amine as confirmed by contact angle measurements of surface wettability. Attachment of polymeriziable end groups to CS and attachment of CS to functionalized substrates demonstrated here are the first steps towards synthesis of CS bottle brush PG mimics.
Assuntos
Materiais Biomiméticos/síntese química , Sulfatos de Condroitina/química , Proteoglicanas/síntese química , Animais , Materiais Biomiméticos/análise , Bovinos , Sulfatos de Condroitina/análise , Proteoglicanas/análise , Soroalbumina Bovina/análise , Soroalbumina Bovina/químicaRESUMO
We report on the nanopatterning of double-bond-terminated silane (5-hexenyltrichlorosilane, HTCS) molecules on titania (TiO2) using conductive atomic force microscopy (AFM). The influences of tip electrostatic potential and scanning velocity, relative humidity and of the repeated application of voltage on the topographic height, width, and hydrophilic and hydrophobic contrast of the resultant patterns were investigated. Tip voltage and tip velocity ( v) were applied between -10 V Assuntos
Nanoestruturas/química
, Nanoestruturas/ultraestrutura
, Titânio/química
, Microscopia de Força Atômica
, Estrutura Molecular