A PULSATILE BIOREACTOR FOR CONDITIONING TISSUE ENGINEERED HEART VALVES

Tissue engineered constructs with autologous adult stem cells capable of self-repair and growth are highly desired replacements for diseased heart valves. However, the current approaches have inadequate mechanical properties to withstand in vivo implantation. Therefore, our group hypothesized that an in vitro environment of physiological intra-cardiac pressures and flow will stimulate stem cells to differentiate and remodel valvular scaffold constructs before implantation. The group developed a pneumatic-driven conditioning system (Aim I) consisting of a three-chambered heart valve bioreactor, a pressurized compliance tank, a reservoir tank, one-way valves, pressure-retaining valves, and pressure transducers. The system can be sterilized using conventional autoclaving and ethylene oxide gas. The most novel feature is its ability to accommodate all clinically relevant sizes of stented or stentless biological, mechanical, or tissue engineered substitutes. A tissue derived heart valve substitute was used to test the bioreactor\u27s functional capabilities (Aim II) at 60 beats per minute. The tests resulted in excellent opening and closing of the valve, pulsatile flows reaching 1400 mL per minute, and aortic pressures reaching 100 mmHg. The bioreactor then tested tissue engineered heart valves (Aim III) made from decellularized and lightly cross-linked tissues. Two stentless porcine aortic heart valves were conditioned in the bioreactor for 21 days. The first was seeded with adipose-derived stem cells (valve 1) and the second with aortic endothelial cells (valve 2). The third valve was made of valve-shaped fibrous sheets encasing a spongy collagen scaffold. It was seeded with human bone marrow-derived stem cells (valve 3) and conditioned in the bioreactor for 8 days. After progressive adaptation, valves were tested at 60 beats per minute and 10 mL per stroke. Each experiment also included a static control. The bioreactor created proper closing and opening of the heart valves and allowed for multiple mounting methods. Results indicated successful cell seeding and attachment in valves 1, 2, and 3; noticeable intercellular alignment in valves 2 and 3; and stem cell differentiation in valve 3. Overall, the conditioning system provides a dynamic three-dimensional cell culture setting designed to provide optimal physiological conditions for tissue engineered heart valve development over extended time periods. The group will continue to develop this approach to study multiple aspects of tissue engineered heart valve development and heart valve pathology

Platform Technologies to Advance Clinically Relevant Tissue Engineered Heart Valve Products

Diseased heart valves are commonly replaced by mechanical, bioprosthetic, or allograft heart valves. These replacements provide major improvements in cardiac function and quality of life, but have significant limitations and eventually require surgical replacement within 15-20 years. These risks are particularly prominent in pediatric patients and young adults. The field of tissue engineering and regenerative medicine, which combines scaffolds and cells, holds great promise in developing living replacement heart valves that would self-repair and grow in size along with the growing children. The long-term goal of this project is to generate living, tissue-engineered heart valves from biological scaffolds and autologous stem cells – a goal that hinges on our ability to create tissue devices that withstand mechanical stresses immediately upon implantation without posing risks of immunological rejection. We hypothesized that these valves can be generated by optimal integration of three main factors: acellular heart valve root scaffolds, autologous stem cells, and construct preconditioning in a bioreactor. Furthermore, we hypothesized that the valves would not be generated without advanced bioreactor systems for the development, conditioning, and translation to clinical practice. To reach this goal, we developed integrated platform technologies for complete aortic valve root (AVR) decellularization, stem cell seeding, and dynamic conditioning before implantation. Unique features include universal “no touch” valve-mounting devices, decellularization in a purpose-designed pulsatile perfusion system, and techniques for in vitro re-vitalization with adipose tissue-derived stem cells (hADSCs) followed by progressive conditioning in our heart valve bioreactors. Acellular porcine AVRs seeded with autologous (sheep) ADSCs were implanted in 10 sheep as right ventricle to pulmonary artery shunts with complete clamping of the pulmonary aorta. Results showed perfect decellularization of the entire porcine AVR and almost complete seeding with ADSCs. Bioreactor studies revealed stem cell pre-differentiation into cells resembling valvular interstitial cells as a response to dynamic stimulation. Animal studies with follow-ups to 12 months are ongoing. Novel customized devices and bioreactor systems are vital to the successful development of tissue engineered heart valve products, especially in preparation for clinical translation. Herein is described some of the basic equipment and expertise necessary for the successful development of tissue-engineered cardiovascular products

Systems to Facilitate Adult Stem Cell Seeding and Conditioning of Aortic Heart Valve Scaffolds

The aim of this research was to develop systems capable of evenly coating replacement heart valves with cells and preconditioning the valves with increasing amounts of sheer stress. We hypothesize that this preconditioning will allow the cells to remain attached after the valve is implanted into the blood stream. The cell seeding device (seeder) and valve conditioning system (bioreactor) were developed using SolidWorks and manufactured on campus. LabView was utilized to control and monitor conditions of both systems. Porcine aortic valves were decellularized, sterilized, crosslinked, neutralized, and coated with cell attachment factors. The valves were placed into the seeder and human adipose derived stem cells were added to the system. After undergoing a specified rotation-pause regimen overnight, the valves were placed in the bioreactor under pulmonary pressures for at least two weeks. Analyses of cellular attachment, retention, and viability were performed. Results show good coverage immediately after seeding and cells beginning to spread after overnight attachment. After two weeks in the bioreactor, many cells remained attached to the valve and were further spread and aligned than static controls and initial time points. Overall, the seeder and bioreactor enable an even coating and retention of cells on a replacement heart valve

Pengaruh Kapasitas Sumber Daya Manusia, Pemanfaatan Teknologi Informasi, dan Pengendalian Intern Akuntansi terhadap Nilai Informasi Pelaporan Keuangan Pemerintah Daerah (Studi Kasus pada Dinas Kabupaten Wonogiri)

The quality of information in financial reporting is strongly influenced by the capacity of the human resources that work well in the preparation of government financial reporting. The use of existing information technology have not yet been able to fully utilized, then the implementation of information technology to be futile and increasingly expensive. This study aims to provide empirical evidence of the influence of human resource capacity, utilization of information technology and internal control of financial reporting information terhdap value of local government in the Office of Wonogiri. This research is an empirical study using survey methods. Data were collected by questionnaires. Samples were obtained 48 respondents. Data were analyzed using multiple linear regression analysis. Based on the analysis we can conclude that the human resource capacity, utilization of information technology, accounting and internal control affect the value of the local government financial reporting information in the Office of Wonogiri. This can be shown by each of the variables p value <0.05. So those hypotheses from H1 until H3 have an effect

Self-adjusting tissue holder

Tissue holders that can be used for gripping natural or synthetic heart valves are described. The tissue holder can include a clamping mechanism and a spring and can be self-adjusting with regard to pressure applied to the tissue gripped in the holder. The tissue holder can be removably attached to systems for processing the tissues and can provide completely hands-free processing of a tissue from development or excisement to implantation and/or completion of testing

Preclinical Testing of Living Tissue-Engineered Heart Valves for Pediatric Patients, Challenges and Opportunities

Introduction: Pediatric patients with cardiac congenital diseases require heart valve implants that can grow with their natural somatic increase in size. Current artificial valves perform poorly in children and cannot grow; thus, living-tissue-engineered valves capable of sustaining matrix homeostasis could overcome the current drawbacks of artificial prostheses and minimize the need for repeat surgeries. Materials and Methods: To prepare living-tissue-engineered valves, we produced completely acellular ovine pulmonary valves by perfusion. We then collected autologous adipose tissue, isolated stem cells, and differentiated them into fibroblasts and separately into endothelial cells. We seeded the fibroblasts in the cusp interstitium and onto the root adventitia and the endothelial cells inside the lumen, conditioned the living valves in dedicated pulmonary heart valve bioreactors, and pursued orthotopic implantation of autologous cell-seeded valves with 6 months follow-up. Unseeded valves served as controls. Results: Perfusion decellularization yielded acellular pulmonary valves that were stable, no degradable in vivo, cell friendly and biocompatible, had excellent hemodynamics, were not immunogenic or inflammatory, non thrombogenic, did not calcify in juvenile sheep, and served as substrates for cell repopulation. Autologous adipose-derived stem cells were easy to isolate and differentiate into fibroblasts and endothelial-like cells. Cell-seeded valves exhibited preserved viability after progressive bioreactor conditioning and functioned well in vivo for 6 months. At explantation, the implants and anastomoses were intact, and the valve root was well integrated into host tissues; valve leaflets were unchanged in size, non fibrotic, supple, and functional. Numerous cells positive for a-smooth muscle cell actin were found mostly in the sinus, base, and the fibrosa of the leaflets, and most surfaces were covered by endothelial cells, indicating a strong potential for repopulation of the scaffold. Conclusions: Tissue-engineered living valves can be generated in vitro using the approach described here. The technology is not trivial and can provide numerous challenges and opportunities, which are discussed in detail in this paper. Overall, we concluded that cell seeding did not negatively affect tissue-engineered heart valve (TEHV) performance as they exhibited as good hemodynamic performance as acellular valves in this model. Further understanding of cell fate after implantation and the timeline of repopulation of acellular scaffolds will help us evaluate the translational potential of this technology

Waste Management on Dairy Farms in Costa Rica

This report, prepared for the Empresa de Servicios Publicos de Heredia (ESPH), investigates methods to prevent pollution of water caused by wastewater discharge of dairy farms in Costa Rica. The report describes how to determine the farming qualities necessary to implement clean technologies, including polyethylene anaerobic digesters and best management practices

Shear Stress Quantification in Tissue Engineering Bioreactor Heart Valves: A Computational Approach

: Tissue-engineered heart valves can grow, repair, and remodel after implantation, presenting a more favorable long-term solution compared to mechanical and porcine valves. Achieving functional engineered valve tissue requires the maturation of human cells seeded onto valve scaffolds under favorable growth conditions in bioreactors. The mechanical stress and strain on developing valve tissue caused by different pressure and flow conditions in bioreactors are currently unknown. The aim of this study is to quantify the wall shear stress (WSS) magnitude in heart valve prostheses under different valve geometries and bioreactor flow rates. To achieve this, this study used fluid-structure interaction simulations to obtain the valve's opening geometries during the systolic phase. These geometries were then used in computational fluid dynamics simulations with refined near-wall mesh elements and ranges of prescribed inlet flow rates. The data obtained included histograms and regression curves that characterized the distribution, peak, and median WSS for various flow rates and valve opening configurations. This study also found that the upper region of the valve near the commissures experienced higher WSS magnitudes than the rest of the valve

Independent Control of the Stretch and Stiffness of a Cell Culture Substrate for Mechanobiological Studies

Cells respond to mechanical stimuli, such as stiffness and stretch, in their environment as shown by changes in phenotype, proliferation, and alignment. We have developed the first method to simultaneously apply stiffness and stretch to a cellular environment using the Flexercell stretching device and the stiffness-tuneable hydrogel, polyacrylamide. Functionality and cell viability were verified and measurements of mechanical stimuli were validated. This method will be applied in future research to study heart valve cell mechanobiology in normal and diseased states