Introducing monitoring and automation in cartilage tissue engineering, toward controlled clinical translation

The clinical application of tissue engineered products requires to be tightly connected with the possibility to control the process, assess graft quality and define suitable release criteria for implantation. The aim of this work is to establish techniques to standardize and control the in vitro engineering of cartilage grafts. The work is organized in three sub-projects: first a method to predict cell proliferation capacity was studied, then an in line technique to monitor the draft during in vitro culture was developed and, finally, a culture system for the reproducible production of engineered cartilage was designed and validated. Real-time measurements of human chondrocyte heat production during in vitro proliferation. Isothermal microcalorimetry (IMC) is an on-line, non-destructive and high resolution technique. In this project we aimed to verify the possibility to apply IMC to monitor the metabolic activity of primary human articular chondrocytes (HAC) during their in vitro proliferation. Indeed, currently, many clinically available cell therapy products for the repair of cartilage lesions involve a process of in vitro cell expansion. Establishing a model system able to predict the efficiency of this lengthy, labor-intensive, and challenging to standardize step could have a great potential impact on the manufacturing process. In this study an optimized experimental set up was first established, to reproducible acquire heat flow data; then it was demonstrated that the HAC proliferation within the IMC-based model was similar to proliferation under standard culture conditions, verifying its relevance for simulating the typical cell culture application. Finally, based on the results from 12 independent donors, the possible predictive potential of this technique was assessed. Online monitoring of oxygen as a non-destructive method to quantify cells in engineered 3D tissue constructs. This project aimed at assessing a technique to monitor graft quality during production and/or at release. A quantitative method to monitor the cells number in a 3D construct, based on the on-line measurement of the oxygen consumption in a perfusion based bioreactor system was developed. Oxygen levels dissolved in the medium were monitored on line, by two chemo-optic flow-through micro-oxygen sensors connected at the inlet and the outlet of the bioreactor scaffold chamber. A destructive DNA assay served to quantify the number of cells at the end of the culture. Thus the oxygen consumption per cell could be calculated as the oxygen drop across the perfused constructs at the end of the culture period and the number of cells quantified by DNA. The method developed would allow to non-invasively monitoring in real time the number of chondrocytes on the scaffold. Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing. The aim of this project was to upscale the size of engineered human cartilage grafts. The main aim of this project consisted in the design and prototyping of a direct perfusion bioreactor system, based on fluidodynamic models (realized in collaboration with the Institute for Bioengineering of Catalonia, Spain), able to guarantee homogeneous seeding and culture conditions trough the entire scaffold surface. The system was then validated and the capability to reproducibly support the process of tissue development was tested by histological, biochemical and biomechanical assays. Within the same project the automation of the designed scaled up bioreactor system, thought as a stand alone system, was proposed. A prototype was realized in collaboration with Applikon Biotechnology BV, The Netherlands. The developed system allows to achieve within a closed environment both cell seeding and culture, controlling some important environmental parameters (e.g. temperature, CO2 and O2 tension), coordinating the medium flow and tracking culture development. The system represents a relevant step toward process automation in tissue engineering and, as previously discussed, enhancing the automation level is an important requirement in order to move towards standardized protocols of graft generation for the clinical practice. These techniques will be critical towards a controlled and standardized procedure for clinical implementation of tissue engineering products and will provide the basis for controlled in vitro studies on cartilage development. Indeed the resulting methods have already been integrated in a streamlined, controlled, bioreactor based protocol for the in vitro production of up scaled engineered cartilage drafts. Moreover the techniques described will serve as the foundation for a recently approved Collaborative Project funded by the European Union, having the goal to produce cartilage tissue grafts. In order to reach this goal the research based technologies and processes described in this dissertation will be adapted for GMP compliance and conformance to regulatory guidelines for the production of engineered tissues for clinical use, which will be tested in a clinical trial

THE ROLE OF A SINGLE FATHER IN DEVELOPING AMERICAN FAMILY VALUES REFLECTED IN “JERSEY GIRL” MOVIE BY KEVIN SMITH

Keluarga merupakan suatu lembaga sosial yang terdiri dari beberapa individu yang berinteraksi satu sama lain dan memiliki peran sosial seperti suami, istri, adik, dan kakak. Di Amerika, terdapat berbagai tipe keluarga seperti nuclear family, extended family, dan keluarga orang tua tunggal.Hal ini dapat diteliti dalam film Jersey Girl. Untuk mendapatkan hasil analisis yang baik , digunakan dua metode penelitian yaitu metode penelitian dengan studi kepustakaan dan juga metode pendekatan secara historis dan eksponensial. Dalam film tersebut digambarkan dengan jelas kehidupan sebuah keluarga ayah tunggal di Amerika yang berusaha menjalankan perannya dalam mengembangkan nilai-nilai keluarga. Dan nilai kekeluargaan tersebut juga mempengaruhi hubungan antara anggota keluarga dan kondisi kehidupan sosial keluarga ayah tunggal di Amerika tersebut

Governance: A Soft Revolution with hard Political and Legal Effects

El supuesto básico de este artículo es que la gobernanza señala un alejamiento de los dos pilares del proyecto moderno: la democracia representativa y las instituciones legislativas. La gobernanza, como fenómeno institucional complejo, que va mucho mas allá de la participación, ha desestructurado sensiblemente los dos principales puntos de referencia de la democracia moderna: pueblo y territorio. Su trato inclusivo y abierto no ha impedido el surgimiento de un lado oscuro, hecho de modos exclusivos: un teatro sin publicidad. Desde la perspectiva de las transformaciones este artículo pone en evidencia la emergencia de una normatividad cambiante y fluida, capaz de adaptarse a las especificidades y a la variabilidad de situaciones y procesos, ofuscando inevitablemente la misma primacía de la legislación.The basic assumption of this article is that governance marks a departure from the two pillars of the project of modernity: representative democracy and legislative institutions. Governance, as a complex institutional phenomenon that goes far beyond participation, has significantly deconstructed the two main points of reference of modern democracy, that is, people and territory. Furthermore, its inclusive and open nature has not prevented the emergence of a dark side, made of exclusive modes: a theater without publicity. From the perspective of transformations, this article highlights the emergence of a changing and fluid normativity, one capable of adapting to the specificity and the variability of situations and processes, inevitably eclipsing the primacy of the legislation itself

Who Are Herodotus\u27 Persians?

In analyzing how Herodotus\u27 descriptions of foreign societies reflect Greek assumptions and prejudices, we have sometimes failed to recognize the extent to which he reports persuasive and historically valid information. This is particularly true of the Persians for whom Herodotus appears to have had access to very good sources, especially perhaps among Medes and Persians living in Asia Minor. This paper argues that Herodotus\u27 representation of Persian character and customs and his understanding of the relationship between the king and his subjects is based on genuine native traditions that reflect an internal debate within Persian elites in the aftermath of their war against Greece