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The treatment of bone defects is facing the situation of lacking donations for autotransplantation. As a valid approach, scaffold-based tissue engineering combines the construction of well-defined porous scaffolds with advanced cell culturing technology to guide tissue regeneration. The role for the scaffold is to provide a suitable environment with a sufficient mechanical stiffness, supports for cell attachment, migration, nutrients and metabolite transport and space for cell remodeling and tissue regeneration. The random copolymers poly(L-lactide-co-ɛ-caprolactone) (poly(LLA-co-CL)) and poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) have been successfully incorporated into 3D porous scaffolds to induce specific interactions with cells and direct osteogenic cell differentiation. In this thesis, these scaffolds have been modified in chemical and physical ways to map and understand requirements for bone regeneration. Scaffold functionalities and properties, such as hydrophilicity, stiffness, size/shape, and reproducibility, were studied. The hydrophilicity was varied by adding 3–20 % (w/w) Tween 80 to poly(LLA-co-CL) and poly(LLA-co-DXO) respectively, which resulted in contact angles from 35° to 15°. With 3 % Tween 80, the resultant mechanical and thermal properties were similar to pristine polymer materials. Tween 80 did not significantly influence cell attachment or proliferation but did stimulate the mRNA expression of osteogenetic markers. The surface functionality and mechanical properties were altered by introducing nanodiamond particles (n-DP) into poly(LLA-co-CL) scaffolds by means of surface physisorption or hybrid blending. Scaffold with n-DP physisorbed showed improved cell attachment, differentiation, and bone reformation. Hybrid n-DP/poly(LLA-co-CL) composites were obtained by direct blending of polylactide modified n-DP (n-DP-PLA) with poly(LLA-coCL). The n-DP-PLA was prepared by sodium hydride-mediated anionic polymerization using n-DP as the initiator. Prepared n-DP-PLA could be dispersed homogenously in organic solvents and blended with poly(LLA-coCL) solution. The n-DP-PLA particles were homogenously distributed in the composite material, which significantly improved mechanical properties. For comparison, the addition of benzoquinone-modified n-DP (n-DP-BQ) did not reinforce poly(LLA-co-CL). This indicated the importance of specific surface grafting, which determined different particle-polymer interactions. For the treatment of critical size defects, a large porous poly(LLA-co-CL) scaffold (12.5 mm diameter × 25 mm thickness) was developed and produced by molding and salt-leaching methods. The large porous scaffolds were evaluated in a scaffold-customized perfusion-based bioreactor system. It was obvious that the scaffold could support improved cell distribution and support the stimulation of human mesenchymal stem cell (hMSC) especially with dynamic flow in a bioreactor. To improve the scaffolding technique, a three-dimensional fiber deposition (3DF) technique was employed to build layer-based scaffolds. Poly(LLA-coCL) scaffolds produced by the 3DF method showed enhanced mechanical properties and a homogeneous distribution of human osteoblasts (hOBs) in the scaffolds. Although poly(LLA-co-CL) was thermally degraded, the degradation did not influence the scaffold mechanical properties. Based on the computerized design, a 3DF scaffold of amorphous copolymer poly(LLAco-CL) provides high-precision control and reproducibility. In summary, the design of porous scaffolds is one of the essential factors in tissue engineering as to mimicking the intrinsic extracellular environment. For bone tissue engineering, an optimized scaffold can maintain a contact angle greater than 35 degrees. Pristine or modified n-DP, introduced as an additive by surface physisorption or direct blending, can improve scaffold mechanical properties and cell response. Various sizes of scaffolds can be easily produced by a mold-mediated salt-leaching method. However, when 100 % reproducibility is required, the 3DF method can be used to create customizable scaffolds. / <p>QC 20140929</p>

En l'actualitat, la indústria farmacèutica utilitza la tecnologia basada en el cultiu in vitro de cèl·lules animals per a la producció de compostos d'elevat interès terapèutic i també, com a model biològic per assajar l'activitat de nous fàrmacs. Moltes empreses fan ús d'aquesta tecnologia ja que es tracta del sistema biològic més apropiat per obtenir proteïnes complexes. Tot i així, pel que fa l'ús de cèl·lules animals, existeixen una sèrie de limitacions importants, ja que s'ha comprovat que una gran part de les cèl·lules presents en aquests cultius moren per apoptosi. La principal causa d'aquest tipus de mort és l'esgotament de determinats nutrients essencials o factors de creixement i l'acumulació de metabòlits tòxics per a la cèl·lula. L'apoptosi representa un greu inconvenient a nivell del cultiu in vitro en bioreactors, ja que disminueix dràsticament la viabilitat del cultiu i en conseqüència, la productivitat del bioreactor.En el present treball, s'han utilitzat les eines que proporciona la biologia molecular per tal de modificar les cèl·lules, fent-les més resistents al procés d'apoptosi. L'objectiu final és aconseguir cèl·lules que mantinguin la viabilitat durant més temps, tot i que en el medi de cultiu es donin senyals per iniciar l'apoptosi. D'aquesta manera es milloraria la productivitat i l'eficiència d'aquests processos. En aquesta tesi doctoral s'han proposat dues estratègies d'inhibició genètiques, una que consisteix en inhibir l'actuació de les Caspases, proteïnes claus en el procés d'apoptosi, i l'altra que es centra a nivell del mitocondri, punt on conflueixen els diferents senyals apoptòtics d'origen intracel·lular. Entre els diferents gens assajats, els resultats més positius s'han obtingut amb la proteïna BHRF-1. En canvi, l'expressió de la proteïna P35 no ha permès protegir les cèl·lules enfront l'apoptosi. Gràcies a l'expressió del gen bhrf-1 és possible retardar la mort per apoptosi dels cultius cel·lulars, augmentant així la productivitat del procés, i garantir la supervivència de les cèl·lules sotmeses durant 72 hores a condicions inductores de l'apoptosi, ja sigui per raons accidentals o per situacions de limitació de nutrients o acumulació de metabòlits tòxics.També s'ha observat que després d'un cert període de manteniment de la resembra, les cèl·lules transfectades perden la capacitat de protegir-se contra l'apoptosi, ja que l'expressió del gen antiapoptòtic s'acaba silenciant. Per aquest motiu, es necessari l'ús de vectors que expressin el gen d'interès al llarg del temps. Els resultats obtinguts han demostrat que els vectors bicistrònics són una bona eina per modificar genèticament les cèl·lules animals ja que per una banda, permetent una integració adequada del fragment de DNA clonat i per l'altra que les modificacions genètiques realitzades perdurin al llarg de temps sense la necessitat d'exercir una selecció constant amb antibiòtic, aspecte molt important sobretot quan s'opera en bioreactors durant períodes molt llarg de temps i es treballa amb volums propis d'un bioreactor. En els cultius en perfusió s'ha vist que l'expressió del gen antiapoptòtic bhrf-1 permet reduir considerablement el nombre de cèl·lules mortes en el cultiu, tot mantenint la densitat de cèl·lules viables. Aquest fet és molt important, ja que es pot allargar la durada del cultiu i en conseqüència, augmentar els nivells de producció del procés. També s'ha observat que aquesta disminució de cèl·lules mortes coincideix amb un alentiment del cicle cel·lular dels hibridomes, suggerint que probablement la proteïna BHRF-1 actua directament sobre el cicle cel·lular i no sobre la via mitocondrial, ja que semblaria que, en condicions anòmales (limitació de nutrients), afavoreix l'entrada de les cèl·lules a l'estat de quiescència (fase G0), la qual cosa impediria l'activació de l'apoptosi. / Nowadays, the pharmaceutical industry uses the in vitro animal cell culture technology for the production of biologicals of therapeutic interest and as a model to test the activity of new drugs. It constitutes the most appropriate system to obtain complex proteins with biological activity. Nevertheless, animal cell culture has certain limitations, such as the early termination of cultures when cells activate the apoptotic program. The depletion of essential nutrients and growing factors in the medium, or increased concentrations of toxic metabolites, are common the cause for cell death. Therefore, the activation of apoptosis in bioreactors seriously compromises the productivity of the bioreactor.In this work, hybridoma cells were genetically engineered in order to make them more resistant to apoptosis under cell culture conditions. The goal was to increase viability of cultured cells, even under adverse apoptosis-inducing conditions, in order to improve productivity of such processes. Two different strategies of genetic inhibition were adopted: inhibition of Caspase activity, and overexpression of antiapoptotic members of the Bcl-2 family.From all genes tested, the best results were obtained with the one that expresses the protein BHRF-1. Otherwise, expression of the baculoviral P35 did not protect cells against apoptosis. Cell cultures expressing gene bhrf-1 lengthened its survival period up to 72 hours when submitted to adverse conditions leading to apoptosis. In this way death due to apoptosis was retarded when nutrients were limited or toxic metabolites accumulated in the medium; or when eventual mechanical incidents occurred. Therefore, the efficiency of the process resulted improved. After several passages in culture, transfected cells lost their capacity to protect themselves from apoptosis, which was attributed to the silencing of antiapoptotic gene expression. For this reason, the hybridoma cells were genetically engineered with bicistronic vectors (containing an IRES element) encoding the gene of interest and a selection marker. This strategy permitted stable expression for large passage number and, moreover, long-term expression of the antiapoptotic genes even in absence of selective pressure. This is of especial importance when operating large bioreactors for long operational periods of time.In perfusion cultures, cultures of cells expressing the antiapoptotic gene bhrf-1 exhibited reduced the number of dead cells and maintained, at the same time, the number of viable cells. This is an important achievement because it allows to lengthen the duration of the cell culture and, consequently, to increase productivity. It was also observed that this decrease in the number of dead cells coincided with a slowing down of the cellular cycle of the hybridomas, suggesting that protein BHRF-1 has a direct effect on the cellular cycle rather than in the mitochondrial pathway. It seems that in anomalous conditions (nutrient restriction) this expression facilitates the switch of the cells to a quiescent stage (phase G0), preventing apoptosis.

Microfluidic biosensors and bioreactors based on immobilized biomaterials are described in this dissertation. Photocrosslinkable hydrogel or polymeric microbeads were used as a supporting matrix for immobilizing E.coli or enzymes in a microfluidic device. This dissertation covers a microfluidic bioreactor based on hydrogel-entrapped E.coli, a microfluidic biosensor based on an array of hydrogel-entrapped enzymes, and a microfluidic bioreactor based on microbead-immobilized enzymes. Hydrogel micropatches containing E.coli were fabricated within a microfluidic channel by in-situ photopolymerization. The cells were viable in the hydrogel micropatch and their membranes could be porated by lysating agents. Entrapment of viable cells within hydrogels, followed by lysis, could provide a convenient means for preparing biocatalysts without the need for enzyme extraction and purification. Our results suggested that hydrogel-entrapped cells, immobilized within microfluidic channels, can act as sensors for small molecules and as bioreactors for carrying out reactions. A microfluidic biosensor based on an array of hydrogel-entrapped enzymes could be used to simultaneously detect different concentrations of the same analyte or multiple analyte in real time. The concentration of an enzyme inhibitor could be quantified using the same basic approach. Isolations of the microchannels within different microfluidic channels could eliminate the possibility of cross talk between enzymes. Finally, we characterized microfluidic bioreactors packed with microbead-immobilized enzymes that can carry out sequential, two-step enzyme-catalyzed reactions under flow conditions. The overall efficiency of the reactors depended on the spatial relationship of the two enzymes immobilized on the beads. Digital simulations confirmed the experimental results.

Thesis (Ph.D.)--University of Central Florida, 2009. / Adviser: Debra R. Reinhart. Includes bibliographical references.

Microdroplet microfluidics has gained much interested in the past decade due to its ability to conduct a wide variety of biological and microfluidic experiments with extremely high repeatability on a mass scale. In particular the ability to culture multiple batches of cells by creating microdroplets with a single encapsulated cell and observe their growth overtime allows for specific conditioning of cells. In addition, when conducting co-culture experiment the induction of a certain stimulus may provide observational rare differences in growth that may be characterized by harnessing a single batch of cells out of thousands of samples. This thesis first presents a variety of microdroplet microfluidic devices that use specific techniques to sufficiently produce, synchronize, merge, and analyze microdroplets. Although many of the devices are capable of producing stable droplets and somewhat efficient synchronization, the overall merging efficiency for most passive or active merging methods alone is lacking. Improvements on such methods and the incorporation of multiple merging methods can lead to a higher overall merging efficiency and greater droplet stability. Also, multiple droplet detection methods can be employed to analyze cellular growth under different conditions, while passive or active sorting methods can be used to acquire particular microdroplet samples downstream. The work presented in this thesis entails the characterization and detailed analysis of all aspects of microdroplet microfluidics necessary to adequately produce a microdroplet co-culture device for microbial studies. This includes the incorporation of multiple microdroplet generators for the production of water droplets immersed in oil serving as bio-reactors for cell culture experiments. In addition, multiple microdroplet synchronization devices were tested to sufficiently align multiple trains of droplets for downstream merging using a variety of passive, active, or combination merging methods. In particular, the use of an electric field can cause destabilization of the surfactant surrounding a microdroplet and allow for the formation of a liquid bridge. The formation of this liquid bridge in conjunction with passive merging methods can lead to droplet electrocoalescence. The incorporation of a more uniform electric field that reduces the angle between the droplet dipole moment and E-field can lead to better droplet merging while reducing voltage and frequency requirements observed in previously publications. The testing, observation, and optimization of such aspects of microdroplet microfluidics are crucial for the advancement and production of sound microdroplet culture devices for a variety of applications including the analysis of dangerous pathogenic substances, drug testing or delivery, and genetic studies.

Thesis--Academic Board of Wageningen University and Academic Board of the UNESCO-IHE Institute for Water Education, 2008. / Description based on print version record. Includes bibliographical references.

Acute Liver Failure (ALF) is a devastating ailment with a high mortality rate and limited treatment alternatives. This study presents a methodology for the design and development of a bio-artificial bioreactor to be used in a Bio-Artificial Liver Support System. The system will ultimately be used either to bridge a patient to orthotopic liver transplant (OLT), the only current cure for end stage ALF, or spontaneous recovery. Methods to optimize and visualize the flow and related mass transfer in the BR are presented. The use of magnetic resonance imaging (MRI), scanning electron microscopy (SEM) and simple testing methodology is applied with emphasis on modeling the flow conditions in the BR. The bioreactor (BR) used in the Bio-Artificial Liver Support System (BALSS), currently under-going animal trials at the University of Pretoria, was modeled and simulated for the flow conditions in the device. Two different perfusion steps were modeled including the seeding of hepatocyte cells and later the clinical perfusion step. It was found that the BR geometry was not optimal with “dead spots” and regions of retarded flow. This would restrict the effective transport of nutrients and oxygen to the cells. The different perfusion rates for the seeding and clinical perfusion steps allowed for different velocity contours with cells seeing inconsistent flow patterns and mass transfer gradients. An optimized BR design is suggested and simulated, that effectively reduces the areas of retarded flow (dead spots) and increases the flow speed uniformly through the BR to an order of magnitude similar to that found in the sinusoidal range. The scaffolding volume was also decreased to allow a larger local cell density promoting cell-cell interaction. Finally a summarized design table for the design of a hepatic BR is presented. / Dissertation (MEng (Mechanical))--University of Pretoria, 2008. / Mechanical and Aeronautical Engineering / unrestricted

Articular cartilage is an avascular, aneural tissue that covers the ends of diarthroidal joints. Once damaged by disease or injury, cartilage lacks the ability to self-repair. Generating tissue engineered cartilage is an exciting field that may provide a possible solution to this problem. The purpose of this study is to determine the efficacy of a through-thickness perfusion bioreactor to generate scaffoldree tissue engineered cartilage. The results of the study show that allowing long-term static culture to cell constructs before perfusion increases the efficacy of the bioreactor. Immediate perfusion of cell constructs in the bioreactor is shown to decrease the efficacy to produce scaffoldree constructs with desirable biomechanical and biochemical properties. The results of the study also show possible options in future works that could increase the efficacy of the bioreactor.