Blood-brain barrier in vitro model: A tissue engineering approach and validation

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

Regulation of Bone Marrow Stem Cells through Oscillatory Shear Stresses - A Heart Valve Tissue Engineering Perspective

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range.

Combined Gene Therapy and Functional Tissue Engineering for the Treatment of Osteoarthritis

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

Manufacturing conditioned roughness and wear of biomedical oxide ceramics for all-ceramic knee implants

Background: Ceramic materials are used in a growing proportion of hip joint prostheses due to their wear resistance and biocompatibility properties. However, ceramics have not been applied successfully in total knee joint endoprostheses to date. One reason for this is that with strict surface quality requirements, there are significant challenges with regard to machining. High-toughness bioceramics can only be machined by grinding and polishing processes. The aim of this study was to develop an automated process chain for the manufacturing of an all-ceramic knee implant. Methods: A five-axis machining process was developed for all-ceramic implant components. These components were used in an investigation of the influence of surface conformity on wear behavior under simplified knee joint motion. Results: The implant components showed considerably reduced wear compared to conventional material combinations. Contact area resulting from a variety of component surface shapes, with a variety of levels of surface conformity, greatly influenced wear rate. Conclusions: It is possible to realize an all-ceramic knee endoprosthesis device, with a precise and affordable manufacturing process. The shape accuracy of the component surfaces, as specified by the design and achieved during the manufacturing process, has a substantial influence on the wear behavior of the prosthesis. This result, if corroborated by results with a greater sample size, is likely to influence the design parameters of such devices.

Sistema colaborativo para monitoramento remoto de equipamentos biomédicos para a videolaparoscopia através do framework web-engineering e o padrão MVC sistema vera-3C

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Mecânica, 2015.

Regulation of bone marrow stem cells through oscillatory shear stresses - A heart valve tissue engineering perspective

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range. ^

Development and mechanical testing of a short intramedullary nail for fixation of femoral rotational osteotomy in cerebral palsy patients

Background: Rotational osteotomy is frequently indicated to correct excessive femoral anteversion in cerebral palsy patients. Angled blade plate is the standard fixation device used when performed in the proximal femur, but extensile exposure is required for plate accommodation. The authors developed a short locked intramedullary nail to be applied percutaneously in the fixation of femoral rotational osteotomies in children with cerebral palsy and evaluated its mechanical properties. Methods: The study was divided into three stages. In the first part, a prototype was designed and made based on radiographic measurements of the femoral medullary canal of ten-year-old patients. In the second, synthetic femoral models based on rapid-prototyping of 3D reconstructed images of patients with cerebral palsy were obtained and were employed to adjust the nail prototype to the morphological changes observed in this disease. In the third, rotational osteotomies were simulated using synthetic femoral models stabilized by the nail and by the AO-ASIF fixed-angle blade plate. Mechanical testing was done comparing both devices in bending-compression and torsion. Results: The authors observed proper adaptation of the nail to normal and morphologically altered femoral models, and during the simulated osteotomies. Stiffness in bending-compression was significantly higher in the group fixed by the plate (388.97 +/- 57.25 N/mm) than in that fixed by the nail (268.26 +/- 38.51 N/mm) as torsional relative stiffness was significantly higher in the group fixed by the plate (1.07 +/- 0.36 Nm/degrees) than by the nail (0.35 +/- 0.13 Nm/degrees). Conclusions: Although the device presented adequate design and dimension to fit into the pediatric femur, mechanical tests indicated that the nail was less stable than the blade plate in bending-compression and torsion. This may be a beneficial property, and it can be attributed to the more flexible fixation found in intramedullary devices.

A hybrid-Trefftz formulation for plane elasticity with selective enrichment of the approximations

This work is related to the so-called non-conventional finite element formulations. Essentially, a methodology for the enrichment of the initial approximation which is typical of the meshless methods and based on the clouds concept is introduced in the hybrid-Trefftz formulation for plane elasticity. The formulation presented allows for the approximation and direct enrichment of two independent fields: stresses in the domains and displacements on the boundaries of the elements. Defined by a set of elements and interior boundaries sharing a common node, the cloud notion is employed to select the enrichment support for the approximation fields. The numerical analysis performed reveals an excellent performance of the resulting formulation, characterized by the good approximation ability and a reduced computational effort. Copyright (C) 2009 John Wiley & Sons, Ltd.

Dynamic Imaging in Electrical Impedance Tomography of the Human Chest With Online Transition Matrix Identification

One of the electrical impedance tomography objectives is to estimate the electrical resistivity distribution in a domain based only on electrical potential measurements at its boundary generated by an imposed electrical current distribution into the boundary. One of the methods used in dynamic estimation is the Kalman filter. In biomedical applications, the random walk model is frequently used as evolution model and, under this conditions, poor tracking ability of the extended Kalman filter (EKF) is achieved. An analytically developed evolution model is not feasible at this moment. The paper investigates the identification of the evolution model in parallel to the EKF and updating the evolution model with certain periodicity. The evolution model transition matrix is identified using the history of the estimated resistivity distribution obtained by a sensitivity matrix based algorithm and a Newton-Raphson algorithm. To numerically identify the linear evolution model, the Ibrahim time-domain method is used. The investigation is performed by numerical simulations of a domain with time-varying resistivity and by experimental data collected from the boundary of a human chest during normal breathing. The obtained dynamic resistivity values lie within the expected values for the tissues of a human chest. The EKF results suggest that the tracking ability is significantly improved with this approach.

A Nonstationary Model of Newborn EEG

The detection of seizure in the newborn is a critical aspect of neurological research. Current automatic detection techniques are difficult to assess due to the problems associated with acquiring and labelling newborn electroencephalogram (EEG) data. A realistic model for newborn EEG would allow confident development, assessment and comparison of these detection techniques. This paper presents a model for newborn EEG that accounts for its self-similar and non-stationary nature. The model consists of background and seizure sub-models. The newborn EEG background model is based on the short-time power spectrum with a time-varying power law. The relationship between the fractal dimension and the power law of a power spectrum is utilized for accurate estimation of the short-time power law exponent. The newborn EEG seizure model is based on a well-known time-frequency signal model. This model addresses all significant time-frequency characteristics of newborn EEG seizure which include; multiple components or harmonics, piecewise linear instantaneous frequency laws and harmonic amplitude modulation. Estimates of the parameters of both models are shown to be random and are modelled using the data from a total of 500 background epochs and 204 seizure epochs. The newborn EEG background and seizure models are validated against real newborn EEG data using the correlation coefficient. The results show that the output of the proposed models has a higher correlation with real newborn EEG than currently accepted models (a 10% and 38% improvement for background and seizure models, respectively).

A matching pursuit-based signal complexity measure for the analysis of newborn EEG

This paper presents a new relative measure of signal complexity, referred to here as relative structural complexity, which is based on the matching pursuit (MP) decomposition. By relative, we refer to the fact that this new measure is highly dependent on the decomposition dictionary used by MP. The structural part of the definition points to the fact that this new measure is related to the structure, or composition, of the signal under analysis. After a formal definition, the proposed relative structural complexity measure is used in the analysis of newborn EEG. To do this, firstly, a time-frequency (TF) decomposition dictionary is specifically designed to compactly represent the newborn EEG seizure state using MP. We then show, through the analysis of synthetic and real newborn EEG data, that the relative structural complexity measure can indicate changes in EEG structure as it transitions between the two EEG states; namely seizure and background (non-seizure).

Effect of multiple transverse modes in self-mixing sensors based on vertical-cavity surface-emitting lasers

We investigate the effect of coexisting transverse modes on the operation of self-mixing sensors based on vertical-cavity surface-emitting lasers (VCSELs). The effect of multiple transverse modes on the measurement of displacement and distance were examined by simulation and in laboratory experiment. The simulation model shows that the periodic change in the shape and magnitude of the self-mixing signal with modulation current can be properly explained by the different frequency-modulation coefficients of the respective transverse modes in VCSELs. The simulation results are in excellent agreement with measurements performed on single-mode and multimode VCSELs and on self-mixing sensors based on these VCSELs.

Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells

A method is presented for the direct extraction of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R-3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (

Designs for an asymmetric gradient set and a compact superconducting magnet for neural magnetic resonance imaging

Imaging of the head and neck is the most commonly performed clinical magnetic resonance imaging (MRI) examination [R. G. Evans and J. R. G. Evans, AJR 157, 603 (1991)]. This is usually undertaken in a generalist MRI instrument containing superconducting magnet system capable of imaging all organs. These generalist instruments are large, typically having a bore of 0.9-1.0 m and a length of 1.7-2.5 m and therefore are expensive to site, somewhat claustrophobic to the patient, and offer little access by attending physicians. In this article, we present the design of a compact, superconducting MRI magnet for head and neck imaging that is less than 0.8 m in length and discuss in detail the design of an asymmetric gradient coil set, tailored to the magnet profile. In particular, the introduction of a radio-frequency FM modulation scheme in concert with a gradient sequence allows the epoch of the linear region of the gradient set to be much closer to the end of the gradient structure than was previously possible. Images from a prototype gradient set demonstrate the effectiveness of the designs. (C) 1999 American Institute of Physics. [S0034-6748(99)04910-2].