What is going on in TennIRM research laboratories?
The University of Tennessee Health Science Center provided $880,000 to initiate TennIRM’s research projects. These funds are being used to seed four working groups: (1) Novel 3D Biofabrication Methodologies and Manufacturing for Enhanced Tissue Regeneration and Implantable Devices; (2) Cell Interactions with 3D Bioprinted Vessels – Basic and Translational Approaches; (3) Stem Cell-Enhanced Tissue Regeneration: Engineering of Vascularized Bone/Cartilage Graft from Adipose-Derived Stem Cells; and (4) Gene Editing of Hematopoietic and Cancer Stem-Like Cells.
Novel 3D Biofabrication Methodologies and Manufacturing for Enhanced Tissue Regeneration and Implantable Devices
This group aims to integrate state-of-the-art metallic, polymeric, and hybrid (combination of metal and polymer) additive manufacturing (AM) technologies to establishing novel 3D fabrication methodologies to create successful tissue regenerative platforms and devices. The team has actively been developing three different aspects. The first aspect has been focused on the AM of magnesium-based biodegradable orthopedic devices. To date, we have designed several AM processes and porous structures, to fabricate metallic scaffolds with different pore sizes, mechanical properties, and cell responses to mimic the bone. We have also investigated hydroxyapatite coating of these structures followed by completed geometrical and mechanical studies. Finally, we have conducted preliminary fibroblast and neutrophil interaction studies with these novel, metallic structures. The second aspect is focused on bioresorbable polymer nano-fiber AM for tissue regeneration templates and medical devices. To date, the team has been able to fabricate two, novel, three-dimensional, near-field electrospinning systems (NFES) that provide us the capability of precise production of fibrous three-dimensional structures in custom shapes replicating different tissues and organs. One system is even modified to conduct such fiber placement on a cylindrical mandrel for fabrication of products such as vascular prosthetics. Process validation and produced fiber characterization has been completed. Current work is focused on evaluation of neutrophil response to the NFES polymer structures. The third aspect has been the development of a unique triple polymer bioink to 3D-print scaffolds for musculoskeletal tissue regeneration. For this study, we have studied cellular compatibility, mechanical, swelling, and degradation properties, and drug release profiles of systematically varied formulations. The printed hydrogels possess tunable mechanical properties and have shown a capability of efficient loading and zero-ordered controlled release of hydrophobic therapeutics. In summary, the results to date are exciting, and we look forward to continued collaboration and success in achieving our goals.
Cell Interactions with 3D Bioprinted Vessels – Basic and Translational Approaches
This project’s goal is to study the cell interactions with 3D bioprinted blood vessel by using a system genetic tool, BXD mouse strains which is a unique genetic model that allows the identification of complex genetic traits regulating a biological response. For this purpose, the first step is to develop mouse model of vascular implantation using stem cell derived 3D bioprinted blood vessel. We have established the method of murine adipose tissue derived stem cell (mADSC) culture, propagation, and the assays to collect phenotypes such as regenerative potential of ADSCs. At the same time, we have developed the mouse model of blood vessel implantation by end-to-end anastomosis of infrarenal abdominal aorta of mice (Fig. 1). This truly unique and new mouse model provides us the chance of investigating the regulation of regeneration of bioprinted blood vessels in the BXD mouse strains. In collaboration with Revotek, the prototype of 3D bioprinter for small vessels has been developed and with a generous charitable grant shipped to Memphis in late June, 2020 (Fig. 2). This 3D bioprinter can print vessel with 1.2 mm outer diameter and 0.5 mm inner diameter as shown in Figure 3. This size closely resembles the size of a human coronary artery. Using the Biosynspherer just arrived to TennIRM, we now have the opportunity to develop customized bioink for use of mouse stem cells to create small blood vessels. We are now working on collecting ADSCs from parent strains of BXD (C57BL/6J and DBA/2J) and comparing the differences on cell surface markers, cell proliferation rate, and cell differentiation potential between strains and sexes. This work will help us to identify critical gene networks that regulate the regeneration of blood vessels. This information will provide novel insights into genetic manipulation and enhancement of 3D bioprinted blood vessels to further increase the success of implantation into human patients.
Mouse surgery of vessel graft implantation. Lower panel shows the blood flow in the implanted graft by ultrasound.
Prototype of 3D bioprinter.
A 3D printed vessel with 1.2mm outer diameter, and 0.5mm inner diameter.
Stem Cell-Enhanced Tissue Regeneration: Engineering of Vascularized Bone/Cartilage Graft from Adipose-Derived Stem Cells
The long-range goal of Project 3 is to re-establish the functional integrity of the OA knee joint, including the osteochondral interface, using stem cells for regenerative repair in the rodent models of arthritis. A problem for using mesenchymal stem cells (MSC) for arthritic joint repair is that <1% of the administered cells localize and persist in target tissues. Investigators in Project 3 have shown that by improving the joint environment, MSC persistence in the post-traumatic knee joint can be enhanced. In particular mechanical stress stimulates reactive oxygen species (ROS) which induce tissue inflammation and degradation. Drs. Karen Hasty and Hongsik Cho have shown that intra-articular injection of the mouse PTOA knee joint with ROS-scavenging poly(propylene sulfide) microspheres improves MSC half-life in the joint. Dr. Brand has established criteria for quantitative µCT analyses of bone loss in mouse models by three-dimensional reconstruction, showing that the oral pathogen Porphyromonas gingivalis will cause trabecular bone loss. This loss contributes to cartilage damage and attraction of inflammatory macrophages producing ROS and inflammatory cytokines. Dr. Smith has shown activation of macrophages can be modified using raspberry ketones (RK) decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines through M2 macrophage polarization to facilitate regenerative repair. RK delivered in regeneration membranes may improve outcomes where excess inflammation impedes regenerative repair. Dr. Miranda has contributed to understanding of transcriptional factors operative in mesenchymal stem cells in osteoblast differentiation and critical for bone regeneration. A study of GATA4, a transcriptional factor regulating osteoblast differentiation, in mice where GATA4 was knocked out in limb bud mesenchyme shows the knock-out MSCs have significant reduction in WNT ligands and WNT signalosome components. This proves that the mechanism of decreased MSCs is due to loss of GATA4 and a WNT10B-dependent positive-autoregulatory loop.
Harnessing Hematopoietic and Cancer-Stemlike Cells for Regenerative Medicine
The laboratory of Gabor Tigyi (UTHSC): Cancer stem-like cells (CSC) are a unique and small population of cells within a cancer that can self-renew, invade and also seed tumor metastasis in distant organs away from the primary tumor. The Tigyi group has been studying the mechanisms with which CSC develop resistance to chemo- and radiation-therapy. This research has identified the key role of the growth factor-like lipid mediator lysophosphatidic acid (LPA) in metastasis and therapy resistance. We have been pursuing a drug discovery research program aimed at developing inhibitors of the LPA type 2 receptor (LPAR2) which we identified plays a central role in ovarian and breast cancer metastasis and therapy resistance. We found that an LPAR2 inhibitor prototype compound designated as AM35 is very effective in killing CSC and enhance the effectiveness when co-applied with radiation and chemotherapy drugs. We also developed small molecule inhibitors of the autotaxin enzyme that generates LPA in carcinoma cells and in the cells of the tumor microenvironment. Two of our autotaxin inhibitors designated BMP-22 and BESA-3 have been found to be particularly effective in blocking the growth of CSC and metastasis in mouse carcinoma models. (Fig. 4).
LPAR2 and autotaxin inhibition disrupts spheroid formation in 4T1 mouse mammary carcinoma stem cells. CSCS form spheroids - mini tumors in culture - which were disrupted by AM35, BMP-22 (ATX inhibitor) or BSEA-3 (mixed ATX/LPA1 inhibitor). Cells were surveyed under 100X magnification (microscopy panels), and spheroids were manually counted, plotted versus AM35 concentration with nanomola