Structure-functionality relationship of collagen scaffolds for tissue engineering

Tissue engineering is a promising technology that enables scientists to create artificial organs or replace damaged tissues using animal cells and other components. For successful tissue regeneration, many factors should be taken into account, however, three components are most crucial: cell, scaffold, and soluble factor(s). In order to check the functionality after regeneration of desired tissues, various approaches have been attempted, depending on the physical, biological, and chemical properties of the tissues. Recently, the importance of the extracellular matrix (ECM) microstructure is being considered to be important in this regard. The ECM is closely associated with various functional properties of the tissues including mechanical properties, diffusivity, and hydraulic conductivity or permeability. Besides providing structural support and determining the physical and functional properties, the ECM plays various roles in tissue physiology by regulating cell morphology, growth and intercellular signaling. The ECM can also be reconfigured by cells during tissue remodeling and wound healing. In this thesis, in order to investigate the structure-functionality relationship of engineered tissues (ETs), computational modeling and experimental studies were performed based on the following three topics: (1) the effect of different ECM structures on the tissue transport property, (2) the effect of the different ECM structures on the cell functionality and subsequent tissue mechanical property, and (3) the evaluation of functionality of new vessel networks formed by modulation of ECM structures. ^ The first study developed computational models (i.e., parameter- and image-based models) using experimental data to predict transport properties (i.e.,permeability and diffusivity) of two different microstructural matrices (i.e., monomer and oligomer) for tissue functionality. The developed computational models underestimated the permeability result compared to what was obtained experimentally. The image- and parameter-based models developed in the present study were able to predict values closest to the experiment data, when compared with previously reported models of permeability. For diffusivity, the computational results showed a similar trend and magnitude to the experimental ones. ^ During cryopreservation of tissues, freezing-induced structural deformation of the tissues and cells occurs due to formation of ice within the intracellular and extracellular spaces. Several studies focused on developing optimal combinations of cryoprotective agent (CPA) and freeze/thaw (F/T) protocols for functional tissue and cell preservation. In the second study, a hypothesis was tested that the modulation of the cytoskeletal structure can mitigate the freezing-induced changes of the functionality, therefore, may reduce the amount of CPA necessary to preserve the tissue\u27s functionality during cryopreservation. In order to test the above hypothesis, the engineered tissues (ETs) were exposed to various F/T conditions with or without CPAs, and the freezing-induced cell-fluid-matrix interactions and subsequent functional properties of the ETs were assessed. Our result showed that, the use of only a small concentration of CPA was very successful in completely preserving the elastic modulus and the viscous friction to the state of the unfrozen 3D stressed structure (STR). This result underscores the importance of CPA in preserving the cytoskeleton structure and how that impacts functional properties of the tissue after freeze-thaw cycles. ^ The third study performed the parametric study to estimate endothelium hydraulic conductivity for vessel functionality. Currently, it is known that formation of vasculatures within the tissues is the most difficult aspect of tissue engineering. Moreover, a method to evaluate new vessel functionality has not been well-established to date. Therefore, a new method with the osmotic pressure-driven vessel deformation and the poroelastic theory was developed using new vessel networks formed by vasculogenesis for hydraulic conductivity estimation. Results showed that the hydraulic conductivity was more sensitive to the elastic modulus compared to other parameters. When the elastic modulus with 10 - 100 Pa and Possions\u27s ratio with 0.3 were applied, the hydraulic conductivity was well-matched with the previously reported hydraulic conductivity

Temperature and heat stress of vessels during cold perfusion of kidney

Paper presented to the 10th International Conference on Heat Transfer, Fluid Mechanics and Thermodynamics, Florida, 14-16 July 2014.The first step of organ cryopreservation is a cryoprotectant perfusion, which can speed cooling. The temperature of the kidney decreased from 37℃ to about 0℃ by perfusion. During the kidney perfusion process, the cryoprotectant enters the kidneys through the renal artery and leaves the kidneys through the renal vein after passing through a series of capillaries. In cold perfusion vessels with a diameter larger than 0.3 mm must be treated individually. The obvious temperature change in the larger vessels will bring in the displacement causing by the thermal contraction. This paper is dedicated to present a comprehensive investigation on the thermal effects of larger blood vessels during cold perfusion including temperature change and corresponding heat stress. A structural model of heat transfer in kidney is developed using currently available anatomical and physiological data. To characterize the effect of thermally significant blood vessels on heat transfer inside the tissues during cold perfusion, the cryoprotectant in the blood vessel was controlled by the energy equation and Navier-Stokes equations. The tiny capillaries and its surrounding biological tissue were treated as the porous media following Darcy's Law. The controlling equations were numerically solved by CFD software. The numerical simulation for the coupled transient thermal field and stress field is carried out by sequentially thermal-structural coupled method based on ANSYS to evaluate the stress fields and of deformations which are established in the blood vessel and tissue. The results indicated that the thermal effects of large blood vessels could remarkably affect the temperature distribution of cold perfusion. And the heat stress obvious changed during cooling, especially for the vein. The maximal heat stress occurred at the export of the vein. This position may be the keys to avoid stress injury during perfusion. This paper provides a guideline to optimize the cold perfusion process from the biomechanics effect.cf201

Development of small-scale fluidised bed bioreactor for 3D cell culture

Three-dimensional cell culture has gained significant importance by producing physiologically relevant in vitro models with complex cell-cell and cell-matrix interactions. However, current constructs lack vasculature, efficient mass transport and tend to reproduce static or short-term conditions. The work presented aimed to design a benchtop fluidised bed bioreactor (sFBB) for hydrogel encapsulated cells to generate perfusion for homogenous diffusion of nutrients and, host substantial biomass for long-term evolution of tissue-like structures and “per cell” performance analysis. The sFBB induced consistent fluidisation of hydrogel spheres while maintaining their shape and integrity. Moreover, this system expanded into a multiple parallel units’ setup with equivalent performances enabling simultaneous comparisons. Long term culture of alginate encapsulated hepatoblastoma cells under dynamic environment led to proliferation of highly viable cell spheroids with a 2-fold increase in cellular density over static (27.3 vs 13.4 million cells/mL beads). Upregulation of hepatic phenotype markers (transcription factor C/EBP-α and drug-metabolism CYP3A4) was observed from an early stage in dynamic culture. This environment also affected ERK1/2 signalling pathway, progressively reducing its activation while increasing it in static conditions. Furthermore, culture of primary human mesenchymal stem cells was evaluated. Cell proliferation was not observed but continuous perfusion sustained their viability and undifferentiated phenotype, enabling differentiation into chondrogenic and adipogenic lineages after de-encapsulation. These biological readouts validated the sFBB as a robust dynamic platform and the prototype design was optimised using computer-aided design and computational fluid dynamics, followed by experimental tests. This thesis proved that dynamic environment promoted by fluidisation sustains biomass viability in long-term cell culture and leads 3D cell constructs with physiologically relevant phenotype. Therefore, this bioreactor would constitute a simple and versatile tool to generate in vitro tissue models and test their response to different agents, potentially increasing the complexity of the system by modifying the scaffold or co-culturing relevant cell types

Freezing processes in cell suspensions evaluated using cryomicroscopy

This thesis aims at evaluating the freezing response of three different cell types, Pacific Oyster embryos, Jurkats and Helas, using the technique of cryomicroscopy. The choice of cells was primarily based on supporting ongoing research work at the Bioengineering Laboratory, Department of Mechanical Engineering at Louisiana State University in Baton Rouge. On a secondary basis, the cells were chosen based on their contrasting nature. While Pacific Oyster being a favorite food in USA calls for successful techniques of cryopreservation of their embryos in order to keep up with the growing demand, Jurkat and HeLa are undesired malignant human cells that require successful cryosurgical techniques for their destruction. The fourth chapter of the thesis addresses the freezing experiments performed on Oyster embryos at freezing rates of 5 deg C/min and 10 deg C/min. During these experiments, embryos were investigated for either dehydration (water transport) or intracellular ice formation (IIF). The next two chapters address the freezing experiments performed on Jurkat cells and HeLa cells respectively. Freezing rates ranging from 1 deg C/min to 50 deg C/min were used for these cells. Once dehydration was observed, the cells were examined for their volume shrinkage. A graph of temperature against normalized volume was plotted using the experimental results. The key cell level parameters were: Reference permeability of cell membrane to water (Lpg), apparent activation energy (ELp), inactive cell volume (Vb), and the ratio of surface area for water transport to the volume of intracellular water (SA/WV). The values of ‘Vb’ for the chosen cells were known from earlier literature. The experimental data was fit into the water transport equation, using a numerical model, in order to obtain the values of the unknown cell level parameters i.e. Lpg and ELp. Finally, Generic Optimal Cooling Rate Equation (GOCRE) was used to determine the optimal cooling rate for the chosen variety of cells. Hence, higher freezing rates were used on the cells, which were investigated for IIF. IIF observed using cryomicroscopy, through darkening probably supported the results for optimal freezing rates, obtained using the water transport experiments and subsequent numerical simulations

Cryopreservation of Adipose Derived Adult Stem Cells and Multidimensional Cell Sheets

Human adipose tissue provides a uniquely abundant and accessible source of adult stem cells. In response to chemical, hormonal or structural stimuli, these human adipose-derived adult stem (ASCs) cells can differentiate along multiple lineage pathways, including adipocytes, chondrocytes, myocytes, neurons and osteoblasts. Successful cryopreservation of scientifically and commercially important ASCs would revolutionize the tissue engineering and regenerative medicine industry. In this research, we have investigated the water transport phenomenon during freezing of several passages of adipose derived adult stem cells and generated the membrane permeability parameters in the presence and absence of cryoprotective agents. These measured permeability parameters were then used to find the optimal cooling rates for freezing ASCs. We have also analyzed the individual and interaction effects of four important thermal parameters (cooling rate, hold time, thawing rate and end temperature) on the post-thaw viability of all passages (passage 0 to passage 4) of ASCs. We have then studied the effect of nanoparticles on the water transport response and apoptotic behavior of biological systems during a cryopreservation process. Further, we have investigated the maintenance of differentiation potential of post-freeze huASCs through histochemical staining. In an attempt to eliminate the usage of chemical CPAs (DMSO/glycerol) during cryopreservation, studies were conducted to investigate the possibility of using non toxic polymers, such as PVP, as cryoprotective agents. Flow cytometry analysis was employed to assess the post-thaw viability and apoptotic response of P1 ASCs frozen stored for more than two weeks in different concentrations of PVP. The results suggest that PVP in fact posses excellent cryoprotective properties and produced acceptable viability when compared to the most routinely used cryopreservation media involving DMSO and serum. In another study, cell sheet engineering approach has been applied to generate multi-dimensional cell sheets for tissue engineering using ASCs

VITRIFICATION AND CHORIOALLANTOIC MEMBRANE (CAM) CULTURE OF BOVINE OVARIAN TISSUE

The overall objectives of this thesis were to develop a short-term culture system and to examine the effects of vitrification and short-term culture on the viability of fresh and vitrified bovine ovarian tissue and the follicles within. The first objective was to compare the health and development of preantral follicles in bovine ovarian tissue, as well as the neovascularization of these tissues, subjected to avian chorioallantoic membrane (CAM) culture with the traditional in vitro culture system. We hypothesized that the chorioallantoic membrane (CAM) of the chicken embryo is a more suitable culture system than traditional in vitro culture. Bovine ovaries were retrieved from a local abattoir and cortical pieces (1-2mm3) were randomly assigned to one of the following groups; control (fixed immediately), CAM or in in vitro culture. Ovarian tissue fragments from both groups were removed on D1, D3 and D5 of culture, fixed, sectioned (5μm) and stained with H&E. The numbers of healthy and degenerated follicles, primordial and activated preantral (primary and secondary), and the number of infiltrated bovine and avian blood vessels were determined using standard stereological procedures. All grafts placed on the traumatized CAM demonstrated increased neovascularization over time. The healthy primordial follicle density decreased over time concomitant with an increase in degenerated (primordial and activated preantral) follicles in both treatment groups. Healthy activated preantral follicle density did not differ between the two culture systems at a given time. In CAM group, blood vessel density increased over time (p = 0.015). The second objective of this thesis was to develop a suitable vitrification protocol for bovine ovarian tissue. The viability of bovine ovarian tissue vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryotop and cryovial) was assessed. We hypothesized that during vitrification the higher cooling rate on the cryotop (open vitrification method) will yield better post-thaw viability of bovine ovarian tissue as compared to the cryovial (closed vitrification method). We also hypothesized that trehalose is a superior non-permeating cryoprotectant to sucrose for vitrification of bovine ovarian tissue. The ovarian tissue was fragmented (1-2mm3) and divided into 6 different treatment groups. Tissues were vitrified in TCM199 supplemented with 15% EG, 15% DMSO, 20% calf serum and 0.5M sucrose or trehalose then placed in a cryovial or on a cryotop. After warming, the vitrified tissues were either immediately placed in 10% formalin (control) or on the chorioallantoic membrane of a 10-day old chicken embryo for 5 days. Follicles from control and vitrified tissue were observed under a light microscope for normal morphology and the total, normal and degenerated follicle densities were determined by standard stereological procedures. Sucrose and trehalose did not differ, nor was a difference observed between the cryovial and the cryotop for total, healthy or degenerated follicle density. Proportion of healthy follicles was higher in the control than all treatment tissues grafted to the CAM. All grafts placed on the traumatized CAM demonstrated presence of avian erythrocytes in the blood vessels after 5 days, but no difference was observed for blood vessel density among treatments. Lastly, the cooling rate of bovine ovarian tissue subjected to open and closed system devices for vitrification was evaluated. A thermocouple wire was used to determine the cooling velocity of 1-2mm3 fragments of bovine ovarian tissue placed on a cryotop (open system) or in a sealed cryovial (closed system). The cooling rate of tissues on the cryotop and in the cryovial was 7481±205.9° C/min and 664±26.0° C/min respectively. In conclusion, the CAM supported the bovine ovarian tissue, thus the CAM culture system may be considered an acceptable alternative to traditional in vitro culture system for bovine ovarian tissue. Furthermore, angiogenesis may be an additional indication of ovarian tissue health. The hypotheses of our second study were refuted. Results indicated that sucrose and trehalose, and the cryotop and cryovial were equally effective in vitrifying bovine ovarian tissue

Experimental and computational strategies for enhancing mass transport and cryopreservation of biological tissues

A bioreactor is a large-scale engineered in vitro device that maintains a 3D arrangement of functioning cells for use in various bioengineering applications. The current work is focused on heat and mass transfer issues related to the bioreactor's performance and applications. Firstly, for bioreactors to achieve high functional output, the cells within its 3D tissues constructs must have adequate supplies of nutrients and gases (O2, CO 2 etc). Among these, O2 transport has been a major challenge since regions of hyperoxia and hypoxia can develop. Hence, in the first phase of this work, an O2 transport based computational model is proposed to help simulate the distribution of O2 through the volume of the 3D tissue constructs under various operational conditions. The advantage of such a predictive model is that it can supply preliminary data, helpful for optimizing O2 delivery to the cells. Secondly, the off the shelf availability of the cells and tissues utilized in the bioreactors is maintained mainly through cryopreservation techniques. In the case of large tissues, cryopreservation success is governed by the cryopreservation protocol used. Therefore, in the second phase of this work, a user friendly computational tool able to predict and compare the effectiveness of various cryopreservation protocols is developed. The computational tool's predictions are briefly validated against experimental results to verify its predictive accuracy. The package is designed to offer a cost effective solution for designing protocol's for cryopreserving 3D tissues and tissue equivalent. Thirdly, with specific relevance to the cryopreservation of liver cells and tissues, it was hypothesized that increased aquaporin (AQP) (integral membrane proteins which aid water transport) expressions on the cellular membrane would improve cellular water transport and thereby improve the cryopreservation efficiency. Experimental results showed increased cell viability following cryopreservation of liver tissues equivalents treated for translocation of AQPs to the cellular membrane, thus confirming the hypothesis to be true. Overall, the computational and experimental strategies proposed in the current work would help enhance heat and mass transport to biological tissues, resulting in potential improvement in the performance of bioreactors and other large scale tissue replacement systems

Analysis of a Hyperbolic Heat Transfer Model in Blood-perfused Biological Tissues with Laser Heating

This paper proposes a hyperbolic heat transport model for a homogeneously perfused biological tissue irradiated by a laser beam. In particular, involving two local energy equations, one for the blood vessel and the other for the tissue, a non-Fourier-like heat equation is introduced and solved analytically using the Laplace transform method. The generalized hyperbolic model obtained is reduced to Pennes' heat transport equation in case the thermal delay time is zero and the solution obtained is in accordance with the numerical and experimental data existing in the literature. In addition, the achieved results also show that the effects of thermal relaxation and blood perfusion on temperature distribution are similar; indeed the highest temperature is expected when the delay time IR increases during tissue cooling. Finally, the consequences of the change in the values of the physical parameters characterizing the model are described and the effect of thermal relaxation on the temperature profile in the tissue during and after laser application is investigated