Enhancing all-in-one bioreactors by combining interstitial perfusion, electrical stimulation, on-line monitoring and testing within a single chamber for cardiac constructs

Tissue engineering strategies have been extensively exploited to generate functional cardiac patches. To maintain cardiac functionality in vitro, bioreactors have been designed to provide perfusion and electrical stimulation, alone or combined. However, due to several design limitations the integration of optical systems to assess cardiac maturation level is still missing within these platforms. Here we present a bioreactor culture chamber that provides 3D cardiac constructs with a bidirectional interstitial perfusion and biomimetic electrical stimulation, allowing direct cellular optical monitoring and contractility test. The chamber design was optimized through finite element models to house an innovative scaffold anchoring system to hold and to release it for the evaluation of tissue maturation and functionality by contractility tests. Neonatal rat cardiac fibroblasts subjected to a combined perfusion and electrical stimulation showed positive cell viability over time. Neonatal rat cardiomyocytes were successfully monitored for the entire culture period to assess their functionality. The combination of perfusion and electrical stimulation enhanced patch maturation, as evidenced by the higher contractility, the enhanced beating properties and the increased level of cardiac protein expression. This new multifunctional bioreactor provides a relevant biomimetic environment allowing for independently culturing, real-time monitoring and testing up to 18 separated patches

A parameterised mathematical model to elucidate osteoblast cell growth in a phosphate-glass microcarrier culture

Tissue engineering has the potential to augment bone grafting. Employing microcarriers as cell-expansion vehicles is a promising bottom-up bone tissue engineering strategy. Here we propose a collaborative approach between experimental work and mathematical modelling to develop protocols for growing microcarrier-based engineered constructs of clinically relevant size. Experiments in 96-well plates characterise cell growth with the model human cell line MG-63 using four phosphate glass microcarrier materials. Three of the materials are doped with 5 mol% TiO2 and contain 0%, 2% or 5% CoO, and the fourth material is doped only with 7% TiO2 (0% CoO). A mathematical model of cell growth is parameterised by finding material-specific growth coefficients through data-fitting against these experiments. The parameterised mathematical model offers more insight into the material performance by comparing culture outcome against clinically relevant criteria: maximising final cell number starting with the lowest cell number in the shortest time frame. Based on this analysis, material 7% TiO2 is identified as the most promising

VAD in failing Fontan: simulation of ventricular, cavo-pulmonary and biventricular assistance in systolic/diastolic ventricular dysfunction and in pulmonary vascular resistance increase.

Aim: Due to the lack of donors, VADs could be an alternative to heart transplantation for Failing Fontan patients (PTs). Considering the complex physiopathology and the type of VAD connection, a numerical model (NM) could be useful to support clinical decisions. The aim of this work is to test a NM simulating the VADs effects on failing Fontan for systolic dysfunction (SD), diastolic dysfunction (DD) and pulmonary vascular resistance increase (PRI). Methods: Data of 10 Fontan PTs were used to simulate the PTs baseline using a dedicated NM. Then, for each PTs a SD, a DD and a PRI were simulated. Finally, for each PT and for each pathology, the VADs implantation was simulated. Results: NM can well reproduce PTs baseline. In the case of SD, LVAD increases the cardiac output (CO) (35%) and the arterial systemic pressure (ASP) (25%). With cavo-pulmonary assistance (RVAD) a decrease of inferior vena cava pressure (IVCP) (39%) was observed with 34% increase of CO. With the BIVAD an increase of ASP (29%) and CO (37%) was observed. In the case of DD, the LVAD increases CO (42%), the RVAD decreases the IVCP. In the case of PRI, the highest CO (50%) and ASP (28%) increase is obtained with an RVAD together with the highest decrease of IVCP (53%). Conclusions: The use of NM could be helpful in this innovative field to evaluate the VADs implantation effects on specific PT to support PT and VAD selection

Biomimetic approach to cardiac tissue engineering

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2004."September 2004."Includes bibliographical references.(cont.) biochemical and morphological properties in the pretreated group. Finally, in order to mimic capillary structure cardiac fibroblasts and myocytes were co-cultured on a scaffold with a parallel channel array that was perfused with culture medium supplemented with synthetic oxygen carrier (PFC emulsion). Presence of the PFC emulsion resulted in significantly higher cell density and improved contractile properties compared to the constructs cultivated in the culture medium alone, by increasing total oxygen content and effective diffusivity.Heart disease is the leading cause of death in the Western world. Tissue engineering may offer alternative treatment options or suitable models for studies of normal and pathological cardiac tissue function in vitro. Current tissue engineering approaches have been limited by diffusional oxygen supply, lack of physical stimuli and absence of multiple cell types characteristic of the native myocardium. We hypothesized that functional, clinically sized (1-5 mm thick), compact cardiac constructs with physiologic cell densities can be engineered in vitro by mimicking cell microenvironment present in the native myocardium in vivo. Since cardiac myocytes have limited ability to proliferate we developed methods of seeding cells at high densities while maintaining cell viability. Cultivation of cardiac constructs in the presence of convective-diffusive oxygen transport in perfusion bioreactors, maintained aerobic cell metabolism, viability and uniform distribution of cells expressing cardiac markers. To improve cell morphology and tissue assembly cardiac constructs were cultivated with electrical stimulation of contraction in a physiologically relevant regime. Electrical stimulation enabled formation of tissue with elongated cells aligned in parallel and with organized ultrastructure remarkably similar to the one present in the native heart. To investigate the effect of multiple cell types on the properties of engineered cardiac tissue cardiac fibroblasts and cardiac myocytes were cultivated synchronously, separately or serially (pretreatment of scaffolds with fibroblasts followed by the addition of myocytes). Presence of fibroblasts remarkably improved contractile response of the engineered cardiac constructs with the superiorby Milica Radisic.Ph.D

The development and validation of a hydrostatic pressure bioreactor for applications in bone tissue engineering

Current orthopaedics treatments of bone defects often involve the use of implanted fixatives and/or autograft procedures to restore function to the afflicted area following injury. Fixatives and implants are usually temporary solutions, since they are intrinsically prone to failure. In addition to this, replacing implants involve expensive and invasive procedures that cause great hardship to patients. Whilst autografts can provide an excellent outcome in healing of the initial injury site, donor site morbidity from the autologous bone graft can lead to complications such as infection, chronic pain and an abnormal walking gait. Bone tissue engineering is a field of science aiming to address these limitations by providing in vitro manufactured bone to replace autografts, and also limit the use of temporary fixatives. Hydrostatic force bioreactors are currently being developed within this field to attempt improve the outcome of the tissue engineered bone by mimicking the forces typically experienced by cells in the native bone niche. Based on this principle, it is hoped that such systems will aid the translation of research in bone tissue engineering from the lab to the clinic. This research aims to investigate and validate the use of a hydrostatic force bioreactor for improving the outcome of in vitro manufactured bone using a clinically relative strategy employing human mesenchymal stem cells seeded in 3D scaffolds. The research first describes a validation process to determine the initial response of cells to hydrostatic pressure in monolayer cultures. The outcome of this study indicated that mechanical responsiveness in cells can vary according to cell phenotype and the integrity of the f-actin cytoskeleton. Next it was demonstrated that hydrostatic pressure can improve the outcome of in vitro bone formation by MG-63 human osteoblast like cells, validating the bioreactor as a potential preconditioning platform. Following this, a model of bone formation in hMSCs/collagen scaffolds was described, whereby a predictable rate of bone formation was determined by adjusting cellular distribution and protein concentration in collagen type-1 scaffolds. Finally, an organotypic fracture repair model was established using explanted embryonic chick femurs to test the hypothesis that hydrostatic preconditioning of hMSC/collagen hydrogels can improve the outcome of fracture repair. The results of this study showed that bioreactor stimulation could enhance the outcome of repair using a combination of undifferentiated hMSC/collagen type-1 scaffolds, and global mechanical signalling (stimulation of entire femur constructs). It was then shown that hydrostatic preconditioning of hMSC seeded hydrogels prior to implantation did not increase the rate of in vitro bone formation. Following implantation of the hydrogels into the fracture repair model, it was demonstrated that highly mineralised preconditioned implants actually inhibited the fracture repair process. In addition to this, it was shown that preconditioned implants with a lower level of mineralisation allowed invasion and bone formation by native cells from the host tissue. Collectively, the results implied that the outcome of repair using this model relied on three main factors: the presence of global hydrostatic stimulation; the lineage commitment of hMSCs in collagen scaffolds at the time of implantation; and the permeability and cell invasion capacity of the implant