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.

In vivo tissue diagnosis for myocardial infarction using optical spectroscopy with novel spectral interpretation algorithms

In recent decades, the rapid development of optical spectroscopy for tissue diagnosis has been indicative of its high clinical value. The goal of this research is to prove the feasibility of using diffuse reflectance spectroscopy and fluorescence spectroscopy to assess myocardial infarction (MI) in vivo. The proposed optical technique was designed to be an intra-operative guidance tool that can provide useful information about the condition of an infarct for surgeons and researchers. ^ In order to gain insight into the pathophysiological characteristics of an infarct, two novel spectral analysis algorithms were developed to interpret diffuse reflectance spectra. The algorithms were developed based on the unique absorption properties of hemoglobin for the purpose of retrieving regional hemoglobin oxygenation saturation and concentration data in tissue from diffuse reflectance spectra. The algorithms were evaluated and validated using simulated data and actual experimental data. ^ Finally, the hypothesis of the study was validated using a rabbit model of MI. The mechanism by which the MI was induced was the ligation of a major coronary artery of the left ventricle. Three to four weeks after the MI was induced, the extent of myocardial tissue injury and the evolution of the wound healing process were investigated using the proposed spectroscopic methodology as well as histology. The correlations between spectral alterations and histopathological features of the MI were analyzed statistically. ^ The results of this PhD study demonstrate the applicability of the proposed optical methodology for assessing myocardial tissue damage induced by MI in vivo. The results of the spectral analysis suggest that connective tissue proliferation induced by MI significantly alter the characteristics of diffuse reflectance and fluorescence spectra. The magnitudes of the alterations could be quantitatively related to the severity and extensiveness of connective tissue proliferation.^

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.

Enhanced anchorage of tissue-engineered cartilage using an osteoinductive approach

Articular cartilage injuries occur frequently in the knee joint. Several methods have been implemented clinically, to treat osteochondral defects but none have been able to produce a long term, durable solution. Photopolymerizable cartilage tissue engineering approaches appear promising; however, fundamentally, forming a stable interface between the tissue engineered cartilage and native tissue, mainly subchondral bone and native cartilage, remains a major challenge. The overall objective of this research is to find a solution for the current problem of dislodgment of tissue engineered cartilage at the defect site for the treatment of degraded cartilage that has been caused due to knee injuries or because of mild to moderate level of osteoarthritis. For this, an in-vitro model was created to analyze the integration of tissue engineered cartilage with the bone, healthy and diseased cartilage over time. We investigated the utility of hydroxyapatite (HA) nanoparticles to promote controlled bone-growth across the bone-cartilage interface in an in vitro engineered tissue model system using bone marrow derived stem cells. We also investigated the application of HA nanoparticles to promote enhance integration between tissue engineered cartilage and native cartilage both in healthy and diseased states. Samples incorporated with HA demonstrated significantly higher interfacial shear strength (at the junction between engineered cartilage and engineered bone and also with diseased cartilage) compared to the constructs without HA (p < 0.05), after 28 days of culture. These findings indicate that the incorporation of HA nanoparticles permits more stable anchorage of the injectable hydrogel-based engineered cartilage construct via augmented integration between bone and cartilage.^

Fiber Scaffolds of Poly (glycerol-dodecanedioate) and its Derivative via Electrospinning for Neural Tissue Engineering

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.

Fiber scaffolds of poly (glycerol-dodecanedioate) and its derivative via electrospinning for neural tissue engineering

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.^

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. ^

In vivo characterization of myocardial tissue post myocardial infarction using combined fluorescence and diffuse reflectance spectroscopy

Accurately assessing the extent of myocardial tissue injury induced by Myocardial infarction (MI) is critical to the planning and optimization of MI patient management. With this in mind, this study investigated the feasibility of using combined fluorescence and diffuse reflectance spectroscopy to characterize a myocardial infarct at the different stages of its development. An animal study was conducted using twenty male Sprague-Dawley rats with MI. In vivo fluorescence spectra at 337 nm excitation and diffuse reflectance between 400 nm and 900 nm were measured from the heart using a portable fiber-optic spectroscopic system. Spectral acquisition was performed on (1) the normal heart region; (2) the region immediately surrounding the infarct; and (3) the infarcted region—one, two, three and four weeks into MI development. The spectral data were divided into six subgroups according to the histopathological features associated with various degrees/severities of myocardial tissue injury as well as various stages of myocardial tissue remodeling, post infarction. Various data processing and analysis techniques were employed to recognize the representative spectral features corresponding to various histopathological features associated with myocardial infarction. The identified spectral features were utilized in discriminant analysis to further evaluate their effectiveness in classifying tissue injuries induced by MI. In this study, it was observed that MI induced significant alterations (p < 0.05) in the diffuse reflectance spectra, especially between 450 nm and 600 nm, from myocardial tissue within the infarcted and surrounding regions. In addition, MI induced a significant elevation in fluorescence intensities at 400 and 460 nm from the myocardial tissue from the same regions. The extent of these spectral alterations was related to the duration of the infarction. Using the spectral features identified, an effective tissue injury classification algorithm was developed which produced a satisfactory overall classification result (87.8%). The findings of this research support the concept that optical spectroscopy represents a useful tool to non-invasively determine the in vivo pathophysiological features of a myocardial infarct and its surrounding tissue, thereby providing valuable real-time feedback to surgeons during various surgical interventions for MI.

Design and performance assessment of aortic heart valve for tissue engineering

Current artificial heart valves are classified as mechanical and bioprosthetic. An appealing pathway that promises to overcome the shortcomings of commercially available heart valves is offered by the interdisciplinary approach of cardiovascular tissue engineering. However, the mechanical properties of the Tissue Engineering Heart Valves (TEHV) are limited and generally fail in the long-term use. To meet this performance challenge novel biodegradable triblock copolymer poly(ethylene oxide)-polypropylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO or F108) crosslinked to Silk Fibroin (F108-SilkC) to be used as tri-leaflet heart valve material was investigated. ^ Synthesis of ten polymers with varying concentration and thickness (55 µm, 75 µm and 100 µm) was achieved via a covalent crosslinking scheme using bifunctional polyethylene glycol diglycidyl ether (PEGDE). Static and fatigue testing were used to assess mechanical properties of films, and hydrodynamic testing was performed to determine performance under a simulated left ventricular flow regime. The crosslinked copolymer (F108-Silk C) showed greater flexibility and resilience, but inferior ultimate tensile strength, by increasing concentration of PEGDE. Concentration molar ratio of 80:1 (F108: Silk) and thickness of 75 µm showed longer fatigue life for both tension-tension and bending fatigue tests. Four valves out of twelve designed satisfactorily complied with minimum performance requirement ISO 5840, 2005. ^ In conclusion, it was demonstrated that the applicability of a degradable polymer in conjugation with silk fibroin for tissue engineering cardiovascular use, specifically for aortic valve leaflet design, met the performance demands. Thinner thicknesses (t<75 µm) in conjunction with stiffness lower than 320 MPa (80:1, F108: Silk) are essential for the correct functionality of proposed heart valve biomaterial F108-SilkC. Fatigue tests were demonstrated to be a useful tool to characterize biomaterials that undergo cyclic loading. ^

In vivo characterization of epileptic tissue with optical spectroscopy

For children with intractable seizures, surgical removal of epileptic foci, if identifiable and feasible, can be an effective way to reduce or eliminate seizures. The success of this type of surgery strongly hinges upon the ability to identify and demarcate those epileptic foci. The ultimate goal of this research project is to develop an effective technology for detection of unique in vivo pathophysiological characteristics of epileptic cortex and, subsequently, to use this technology to guide epilepsy surgery intraoperatively. In this PhD dissertation the feasibility of using optical spectroscopy to identify uniquein vivo pathophysiological characteristics of epileptic cortex was evaluated and proven using the data collected from children undergoing epilepsy surgery. ^ In this first in vivo human study, static diffuse reflectance and fluorescence spectra were measured from the epileptic cortex, defined by intraoperative ECoG, and its surrounding tissue from pediatric patients undergoing epilepsy surgery. When feasible, biopsy samples were taken from the investigated sites for the subsequent histological analysis. Using the histological data as the gold standard, spectral data was analyzed with statistical tools. The results of the analysis show that static diffuse reflectance spectroscopy and its combination with static fluorescence spectroscopy can be used to effectively differentiate between epileptic cortex with histopathological abnormalities and normal cortex in vivo with a high degree of accuracy. ^ To maximize the efficiency of optical spectroscopy in detecting and localizing epileptic cortex intraoperatively, the static system was upgraded to investigate histopathological abnormalities deep within the epileptic cortex, as well as to detect unique temporal pathophysiological characteristics of epileptic cortex. Detection of deep abnormalities within the epileptic cortex prompted a redesign of the fiberoptic probe. A mechanical probe holder was also designed and constructed to maintain the probe contact pressure and contact point during the time dependent measurements. The dynamic diffuse reflectance spectroscopy system was used to characterize in vivo pediatric epileptic cortex. The results of the study show that some unique wavelength dependent temporal characteristics (e.g., multiple horizontal bands in the correlation coefficient map γ(λref = 800 nm, λcomp ,t)) can be found in the time dependent recordings of diffuse reflectance spectra from epileptic cortex defined by ECoG.^