Tissue engineering (TE) approaches strive to regenerate or replace an organ or tissue. The successful development and subsequent integration of a TE construct is contingent on a series of in vitro and in vivo events that result in an optimal construct for implantation. Current widely used methods for evaluation of constructs are incapable of providing an accurate compositional assessment without destruction of the construct. In this review, we discuss the contributions of vibrational spectroscopic assessment for evaluation of tissue engineered construct composition, both during development and post-implantation. Fourier transform infrared (FTIR) spectroscopy in the mid and near-infrared range, as well as Raman spectroscopy, are intrinsically label free, can be non-destructive, and provide specific information on the chemical composition of tissues. Overall, we examine the contribution that vibrational spectroscopy via fiber optics and imaging have to tissue engineering approaches.
Objective Articular cartilage exists in a hypoxic environment, which motivates the use of hypoxia-simulating chemical agents to improve matrix production in cartilage tissue engineering. The aim of this study was to investigate whether dimethyloxalylglycine (DMOG), a HIF-1α stabilizer, would improve matrix production in 3-dimensional (3D) porcine synovial-derived mesenchymal stem cell (SYN-MSC) co-culture with chondrocytes. Design Pellet cultures and scaffold-based engineered cartilage were grown in vitro to determine the impact of chemically simulated hypoxia on 2 types of 3D cell culture. DMOG-treated groups were exposed to DMOG from day 14 to day 21 and grown up to 6 weeks with n = 3 per condition and time point. Results The addition of DMOG resulted in HIF-1α stabilization in the exterior of the engineered constructs, which resulted in increased regional type II collagen deposition, but the stabilization did not translate to overall increased extracellular matrix deposition. There was no increase in HIF-1α stabilization in the pellet cultures. DMOG treatment also negatively affected the mechanical competency of the engineered cartilage. Conclusions Despite previous studies that demonstrated the efficacy of DMOG, here, short-term treatment with DMOG did not have a uniformly positive impact on the chondrogenic capacity of SYN-MSCs in either pellet culture or in scaffold-based engineered cartilage, as evidenced by reduced matrix production. Such 3D constructs generally have a naturally occurring hypoxic center, which allows for the stabilization of HIF-1α in the interior tissue. Thus, short-term addition of DMOG may not further improve this in cartilage tissue engineered constructs.
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Decellularization protocols for both whole organs and tissue parts have been developed and successful for many organs but not for the thymus. The decellularization process maintains the integrity of the organ by preserving the components of the extracellular matrix, including proteins such as collagen and fibronectin. This allows for an acellular, three dimensional biological scaffold of the desired organ which can then be repopulated with cells. Routine decellularization techniques published for other tissue such as the heart and the lung were inadequate for the thymus and ablated the extracellular matrix. Novel techniques were discovered. The aim of this project is to apply the idea of decellularization to the thymus, using thymic tissue procured from murine, porcine, and human models. Thymic tissue has been decellularized using two methods; SDS (sodium dodecyl sulfate) based protocols and by Deoxycholic Acid/Triton-X mixture protocols, and examined for remnant cells and extracellular proteins through histological staining and SEM imaging. We have confirmed complete decelluarization with murine thymic tissue and are advancing to porcine and human samples. We have identified varying decelluarization techniques that provide multiple degrees of integrity of the extra cellular matrix. These techniques may be used in tissue engineering strategies to repopulate a thymic construct for thymic reconstitution.
Tissue engineering of cartilage for tissue repair has many challenges, including the inability to assess when the developing construct has reached compositional maturity for implantation. The goal of this study...
Microfluidic systems have enormous potential as investigational devices in biomedical research, in particular immunology. Microfluidic devices can be fabricated with the precise regulation of any number of parameters including controlled surface chemistries, geometrical dimensions, signal input and output and timing. The adjustable nature of microfluidic devices makes them an ideal platform to recreate cellular microenvironments for studying cell-cell interactions, migration, antibody and cytokine production, and differentiation in vitro. Investigations with customized microfluidic devices have the potential for elucidating the mechanism behind T lymphocyte commitment to either the αβ or γδ lineage. Both types of T cells arise from immature CD4-CD8- precursors in the thymus but diverge during development. Recent publications indicate that γδ T cell receptor (TCR) works in concert with ligand to direct progenitors to the γδ fate. We propose to develop a microfluidic device in which immature T lymphocytes can migrate through a thymus-like microenvironment allowing for αβ or γδ lineage commitment. As of yet, the microfluidic device fabrication protocol has been developed and preliminary cell culture experiments with the commonly used murine stromal cell line OP9 have success. Such a device would allow for analysis of specific developmental points in T cell development.
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