Intrinsically disordered proteins access a range of hysteretic phase separation behaviors.

The phase separation behavior of intrinsically disordered proteins (IDPs) is thought of as analogous to that of polymers that undergo equilibrium lower or upper critical solution temperature (LCST and UCST, respectively) phase transition. This view, however, ignores possible nonequilibrium properties of protein assemblies. Here, by studying IDP polymers (IDPPs) composed of repeat motifs that encode LCST or UCST phase behavior, we discovered that IDPs can access a wide spectrum of nonequilibrium, hysteretic phase behaviors. Experimentally and through simulations, we show that hysteresis in IDPPs is tunable and that it emerges through increasingly stable interchain interactions in the insoluble phase. To explore the utility of hysteretic IDPPs, we engineer self-assembling nanostructures with tunable stability. These findings shine light on the rich phase separation behavior of IDPs and illustrate hysteresis as a design parameter to program nonequilibrium phase behavior in self-assembling materials.

Micellar Self-Assembly of Recombinant Resilin-/Elastin-Like Block Copolypeptides.

Reported here is the synthesis of perfectly sequence defined, monodisperse diblock copolypeptides of hydrophilic elastin-like and hydrophobic resilin-like polypeptide blocks and characterization of their self-assembly as a function of structural parameters by light scattering, cryo-TEM, and small-angle neutron scattering. A subset of these diblock copolypeptides exhibit lower critical solution temperature and upper critical solution temperature phase behavior and self-assemble into spherical or cylindrical micelles. Their morphologies are dictated by their chain length, degree of hydrophilicity, and hydrophilic weight fraction of the ELP block. We find that (1) independent of the length of the corona-forming ELP block there is a minimum threshold in the length of the RLP block below which self-assembly does not occur, but that once that threshold is crossed, (2) the RLP block length is a unique molecular parameter to independently tune self-assembly and (3) increasing the hydrophobicity of the corona-forming ELP drives a transition from spherical to cylindrical morphology. Unlike the self-assembly of purely ELP-based block copolymers, the self-assembly of RLP-ELPs can be understood by simple principles of polymer physics relating hydrophilic weight fraction and polymer-polymer and polymer-solvent interactions to micellar morphology, which is important as it provides a route for the de novo design of desired nanoscale morphologies from first principles.

Molecular description of the LCST behavior of an elastin-like polypeptide.

Elastin-like polypeptides (ELPs) with the repeat sequence of VPGVG are widely used as a model system for investigation of lower critical solution temperature (LCST) transition behavior. In this paper, the effect of temperature on the structure, dynamics and association of (VPGVG)18 in aqueous solution is investigated using atomistic molecular dynamics simulations. Our simulations show that as the temperature increases the ELP backbones undergo gradual conformational changes, which are attributed to the formation of more ordered secondary structures such as ß-strands. In addition, increasing temperature changes the hydrophobicity of the ELP by exposure of hydrophobic valine-side chains to the solvent and hiding of proline residues. Based on our simulations, we conclude that the transition behavior of (VPGVG)18 can be attributed to a combination of thermal disruption of the water network that surrounds the polypeptide, reduction of solvent accessible surface area of the polypeptide, and increase in its hydrophobicity. Simulations of the association of two (VPGVG)18 molecules demonstrated that the observed gradual changes in the structural properties of the single polypeptide chain are enough to cause the aggregation of polypeptides above the LCST. These results lead us to propose that the LCST phase behavior of poly(VPGVG) is a collective phenomenon that originates from the correlated gradual changes in single polypeptide structure and the abrupt change in properties of hydration water around the peptide and is a result of a competition between peptide-peptide and peptide-water interactions. This is a computational study of an important intrinsically disordered peptide system that provides an atomic-level description of structural features and interactions that are relevant in the LCST phase behavior.

Protein polymer hydrogels by in situ, rapid and reversible self-gelation.

Protein-based biomaterials are an important class of materials for applications in biotechnology and medicine. The exquisite control of their composition, stereochemistry, and chain length offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here, we report the synthesis of a thermally responsive peptide polymer-based hydrogel composed of a recombinant elastin-like polypeptide (ELP) that rapidly forms a reversibly cross-linked hydrogel by the formation of intermolecular disulfide cross-links. To do so, we designed and synthesized ELPs that incorporate periodic cysteine residues (cELPs), and show that cELPs are thermally responsive protein polymers that display rapid gelation under physiologically relevant, mild oxidative conditions. Gelation of cELPs, at concentrations as low as 2.5 wt%, occurs in ≈ 2.5 min upon addition a low concentration of hydrogen peroxide (0.3 wt%). We show the utility of these hydrogels for the sustained release of a model protein in vitro, and demonstrate the ability of this injectable biomaterial to pervade tumors to maximize tumor coverage and retention time upon intratumoral injection. cELPs represent a new class of injectable reversibly cross-linked hydrogels with properties intermediate between ELP coacervates and chemically cross-linked ELP hydrogels that will find useful applications in drug delivery and tissue engineering.

Reinforced Portland cement porous scaffolds for load-bearing bone tissue engineering applications.

Modified Portland cement porous scaffolds with suitable characteristics for load-bearing bone tissue engineering applications were manufactured by combining the particulate leaching and foaming methods. Non-crosslinked polydimethylsiloxane was evaluated as a potential reinforcing material. The scaffolds presented average porosities between 70 and 80% with mean pore sizes ranging from 300 µm up to 5.0 mm. Non-reinforced scaffolds presented compressive strengths and elastic modulus values of 2.6 and 245 MPa, respectively, whereas reinforced scaffolds exhibited 4.2 and 443 MPa, respectively, an increase of ∼62 and 80%. Portland cement scaffolds supported human osteoblast-like cell adhesion, spreading, and propagation (t = 1-28 days). Cell metabolism and alkaline phosphatase activity were found to be enhanced at longer culture intervals (t ≥ 14 days). These results suggest the possibility of obtaining strong and biocompatible scaffolds for bone repair applications from inexpensive, yet technologically advanced materials such as Portland cement.

PREGUNTAS Y RESPUESTAS SOBRE MEDICINA REGENERATIVA*

La medicina regenerativa busca desarrollar estrategias para promover la regeneración de tejidos dañados. El auge de esta área de investigación biomédica y su potencial para el tratamiento de una gran variedad de enfermedades-como las neurodegenerativas, hepáticas y cardiacas, o quemaduras de piel y otros traumas-, demanda un análisis continuo sobre el estado de las investigaciones actuales, su impacto, lo que se espera en los próximos años y los debates éticos asociados. Para responder éstas y otras preguntas, revisamos algunos de los avances más importantes ocurridos en año 2011 y consultamos la opinión de un experto en medicina regenerativa, el profesor Felipe Prósper Cardoso de la Universidad de Navarra en España. Si bien los avances han sido muy significativos en el laboratorio, especialmente en la derivación de células autólogas (diferenciadas o pluripotentes) y el desarrollo de biomateriales que facilitan y promueven la regeneración, la traslación clínica de las terapias de medicina regenerativa aún es incipiente y constituye un gran reto para investigadores en esta área.

Regenerative Medicine seeks to develop strategies to restore damaged tissues. The recent boom of this research field and its potential for the treatment of a wide variety of diseases -such as neurodegenerative, hepatic and cardiac, or skin burns and various traumas- demands an ongoing analysis of the most recent investigations, their impact, the associated ethical debates, as well as the research horizon in the coming years. To answer these and other questions we reviewed the most important advances reported throughout the year 2011 and consulted the opinion of a regenerative medicine expert, profesor Felipe Prosper Cardoso from the University of Navarra in Spain. Despite the significant progress in the laboratory regarding the derivation of autologous cells (whether differentiated or pluripotent) and the development of biomaterials that facilitate regeneration, the clinical translation of regenerative medicine therapies is still incipient and constitutes a grand challenge for researchers in this fascinating field.

Engineering antibody fragments: replicating the immune system and beyond: [review] / La ingeniería de fragmentos de anticuerpos: imitando y expandiendo el sistema inmune: [revisión]

Since genetic engineering of humanized murine monoclonal antibodies was first demonstrated over two decades ago, antibody engineering technologies have evolved based upon an increasing understanding of the mechanisms involved in antibody generation in vivo, and a constant search for alternative routes to evolve and exploit the characteristics of antibodies. As a result, antibody engineers have devised innovative strategies for the rapid evolution and selection of antibodies and novel antibody designs (i.e., antibody fragments). Phage display, cell display and ribosome display technologies, which comprise the core of the currently available technologies for the discovery and preparation of such antibodies, are reviewed herein. This article intends to communicate the state-of-the-art technology available for the engineering of antibodies to a general readership interested in this important field. Therefore, important immunology concepts are introduced before detailed descriptions of the three antibody engineering technologies are presented in later sections. A comparison of these methodologies suggests that despite the predominance of phage display for the engineering of antibody fragments in the past 20 years, cell display and ribosome display will likely gain importance in the selection and discovery of the antibody fragments in the future. Finally, these technologies are likely to play an important role in the production of the next generation of antibody-based therapeutics.

Las tecnologías para la ingeniería de anticuerpos han evolucionado durante las últimas dos décadas, desde la demostración de la posibilidad de humanizar anticuerpos monoclonales de ratón mediante ingeniería genética, apoyadas en el creciente entendimiento de los mecanismos involucrados en la generación de anticuerpos in vivo, y en una búsqueda constante de rutas alternativas para evolucionar y explotar sus características. Es así como los ingenieros de anticuerpos han desarrollado estrategias innovadoras para la evolución y selección de anticuerpos y de novedosos diseños de anticuerpos conocidos como fragmentos de anticuerpos. Esta revisión se enfoca en tres tecnologías que comprenden el núcleo de las tecnologías actualmente disponibles para el descubrimiento y preparación de tales anticuerpos: la presentación en fagos, la presentación en células, y la presentación en ribosomas. Este artículo busca presentar el estado del arte de estas tecnologías a un grupo general de lectores interesados en este campo, por lo que inicialmente se introducen importantes conceptos de inmunología requeridos para comprender en detalle las tecnologías discutidas. Una comparación de estas metodologías para la ingeniería de anticuerpos sugiere que a pesar del dominio de las tecnologías basadas en la presentación en fagos durante los últimos 20 años, en los próximos años la presentación en células y la presentación en ribosomas probablemente ganarán importancia para la selección y descubrimiento de fragmentos de anticuerpos. Finalmente, es probable que estas tecnologías jueguen un papel importante en la producción de la siguiente generación de terapéuticos basados en anticuerpos.

Engineering antibody fragments: replicating the immune system and beyond / La ingeniería de fragmentos de anticuerpos: imitando y expandiendo el sistema inmune

Since genetic engineering of humanized murine monoclonal antibodies was first demonstrated over two decades ago, antibody engineering technologies have evolved based upon an increasing understanding of the mechanisms involved in antibody generation in vivo, and a constant search for alternative routes to evolve and exploit the characteristics of antibodies. As a result, antibody engineers have devised innovative strategies for the rapid evolution and selection of antibodies and novel antibody designs (i.e., antibody fragments). Phage display, cell display and ribosome display technologies, which comprise the core of the currently available technologies for the discovery and preparation of such antibodies, are reviewed herein. This article intends to communicate the state-of-the-art technology available for the engineering of antibodies to a general readership interested in this important field. Therefore, important immunology concepts are introduced before detailed descriptions of the three antibody engineering technologies are presented in later sections. A comparison of these methodologies suggests that despite the predominance of phage display for the engineering of antibody fragments in the past 20 years, cell display and ribosome display will likely gain importance in the selection and discovery of the antibody fragments in the future. Finally, these technologies are likely to play an important role in the production of the next generation of antibody-based therapeutics.

Las tecnologías para la ingeniería de anticuerpos han evolucionado durante las últimas dos décadas, desde la demostración de la posibilidad de humanizar anticuerpos monoclonales de ratón mediante ingeniería genética, apoyadas en el creciente entendimiento de los mecanismos involucrados en la generación de anticuerpos in vivo, y en una búsqueda constante de rutas alternativas para evolucionar y explotar sus características. Es así como los ingenieros de anticuerpos han desarrollado estrategias innovadoras para la evolución y selección de anticuerpos y de novedosos diseños de anticuerpos conocidos como fragmentos de anticuerpos. Esta revisión se enfoca en tres tecnologías que comprenden el núcleo de las tecnologías actualmente disponibles para el descubrimiento y preparación de tales anticuerpos: la presentación en fagos, la presentación en células, y la presentación en ribosomas. Este artículo busca presentar el estado del arte de estas tecnologías a un grupo general de lectores interesados en este campo, por lo que inicialmente se introducen importantes conceptos de inmunología requeridos para comprender en detalle las tecnologías discutidas. Una comparación de estas metodologías para la ingeniería de anticuerpos sugiere que a pesar del dominio de las tecnologías basadas en la presentación en fagos durante los últimos 20 años, en los próximos años la presentación en células y la presentación en ribosomas probablemente ganarán importancia para la selección y descubrimiento de fragmentos de anticuerpos. Finalmente, es probable que estas tecnologías jueguen un papel importante en la producción de la siguiente generación de terapéuticos basados en anticuerpos.

ISOLATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS AND EVALUATION OF THEIR OSTEOGENIC POTENTIAL

Human bone marrow mesenchymal stem cells (hBMSCs) comprise a cell population capable of self-renewal and multilineage differentiation commonly isolated from bone marrow aspirates of large bones. Their osteogenic potential has been extensively exploited for the biological evaluation of scaffolds or biomaterials with applications in bone tissue engineering. This work aimed to isolate hBMSCs from femoral heads of patients undergoing total hip arthroplasty and to evaluate their osteogenic potential. Briefly, the trabecular bone was extracted and mechanically disaggregated; the released cells were cultured and non-adherent cells were removed after 4 days. The osteogenic potential was evaluated at the fifth passage after 14 and 20 days of induction, comparing cultures with and without osteogenic supplements, via Alizarin red staining and the quantification of the gene expression levels of the osteogenic markers collagen type I, osteonectin and bone sialoprotein through real-time RT-PCR. The obtained hBMSCs presented a stable undifferentiated phenotype after prolonged cell culture, matrix mineralization capabilities and expression of osteoblast phenotype upon osteogenic induction. The three markers were up-regulated in cultures under osteogenic conditions and 2 fold differences in their expression levels were found to be significant for the onset of the differentiation process. The obtained hBMSCs may have applications on the in vitro evaluation of the osteoinductivity of different biomaterials, bioactive molecules or tissue engineering scaffolds.

Las células madre mesenquimatosas de médula ósea humana (abreviadas hBMSCs) constituyen una fuente de células auto-renovables con alto potencial de diferenciación, comúnmente aisladas a partir de los aspirados medulares en huesos largos. Su diferenciación hacia el linaje osteogénico, por ejemplo, ha sido ampliamente utilizada para la evaluación biológica de biomateriales o matrices con aplicaciones en la ingeniería de tejidos óseos. El objetivo de este trabajo consistió en aislar hBMSCs a partir de la cabeza femoral de pacientes sometidos a artroplastia total de cadera, así como evaluar su potencial osteogénico. Brevemente, se extrajo el hueso esponjoso y se disgregó mecánicamente; las células desprendidas se cultivaron y las células no adherentes se eliminaron luego de 4 días. El potencial osteogénico se evaluó en la quinta generación de cultivo, mediante ensayos de diferenciación a 14 y 20 días donde se compararon cultivos con y sin suplementos osteogénicos. La evaluación se realizó mediante tinción con Alizarina Roja y la cuantificación de los niveles de expresión génica de los marcadores osteogénicos colágeno tipo I, osteonectinca y sialoprotiena ósea mediante RT-PCR en tiempo real. Las hBMSCs obtenidas presentaron un fenotipo no-diferenciado estable, así como la capacidad de mineralizar la matriz extracelular y expresar un fenotipo similar al osteoblasto durante la inducción osteogénica. Los tres marcadores evaluados se sobre-expresaron en los cultivos en condiciones osteogénicas, y se encontró que cambios hasta de 2X en sus niveles de expresión son relevantes para el desarrollo del proceso de diferenciación. El modelo de hBMSCS presentado podría ser utilizado para la evaluación in vitro de la osteoinductividad de diferentes biomateriales, moléculas bioactivas o matrices para ingeniería de tejidos.