Lipid Coated Microbubbles and Low Intensity Pulsed Ultrasound Enhance Chondrogenesis of Human Mesenchymal Stem Cells in 3D Printed Scaffolds

Lipid-coated microbubbles are used to enhance ultrasound imaging and drug delivery. Here we apply these microbubbles along with low intensity pulsed ultrasound (LIPUS) for the first time to enhance proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in a 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) hydrogel scaffold. The hMSC proliferation increased up to 40% after 5 days of culture in the presence of 0.5% (v/v) microbubbles and LIPUS in contrast to 18% with LIPUS alone. We systematically varied the acoustic excitation parameters—excitation intensity, frequency and duty cycle—to find 30 mW/cm2, 1.5 MHz and 20% duty cycle to be optimal for hMSC proliferation. A 3-week chondrogenic differentiation results demonstrated that combining LIPUS with microbubbles enhanced glycosaminoglycan (GAG) production by 17% (5% with LIPUS alone), and type II collagen production by 78% (44% by LIPUS alone). Therefore, integrating LIPUS and microbubbles appears to be a promising strategy for enhanced hMSC growth and chondrogenic differentiation, which are critical components for cartilage regeneration. The results offer possibilities of novel applications of microbubbles, already clinically approved for contrast enhanced ultrasound imaging, in tissue engineering

Biomimetic three-dimensional nanocrystalline hydroxyapatite and magnetically synthesized single-walled carbon nanotube chitosan nanocomposite for bone regeneration

Owen Im1, Jian Li2, Mian Wang2, Lijie Grace Zhang2,3, Michael Keidar2,31Department of Biomedical Engineering, Duke University, Durham, NC; 2Department of Mechanical and Aerospace Engineering, 3Institute for Biomedical Engineering and Institute for Nanotechnology, The George Washington University, Washington, DC, USABackground: Many shortcomings exist in the traditional methods of treating bone defects, such as donor tissue shortages for autografts and disease transmission for allografts. The objective of this study was to design a novel three-dimensional nanostructured bone substitute based on magnetically synthesized single-walled carbon nanotubes (SWCNT), biomimetic hydrothermally treated nanocrystalline hydroxyapatite, and a biocompatible hydrogel (chitosan). Both nanocrystalline hydroxyapatite and SWCNT have a biomimetic nanostructure, excellent osteoconductivity, and high potential to improve the load-bearing capacity of hydrogels.Methods: Specifically, three-dimensional porous chitosan scaffolds with different concentrations of nanocrystalline hydroxyapatite and SWCNT were created to support the growth of human osteoblasts (bone-forming cells) using a lyophilization procedure. Two types of SWCNT were synthesized in an arc discharge with a magnetic field (B-SWCNT) and without a magnetic field (N-SWCNT) for improving bone regeneration.Results: Nanocomposites containing magnetically synthesized B-SWCNT had superior cytocompatibility properties when compared with nonmagnetically synthesized N-SWCNT. B-SWCNT have much smaller diameters and are twice as long as their nonmagnetically prepared counterparts, indicating that the dimensions of carbon nanotubes can have a substantial effect on osteoblast attachment.Conclusion: This study demonstrated that a chitosan nanocomposite with both B-SWCNT and 20% nanocrystalline hydroxyapatite could achieve a higher osteoblast density when compared with the other experimental groups, thus making this nanocomposite promising for further exploration for bone regeneration.Keywords: nanomaterials, single-walled carbon nanotube, nanocrystalline hydroxyapatite, chitosan, bone regeneration, biomimeti

Cold atmospheric plasma for selectively ablating metastatic breast cancer cells

Traditional breast cancer treatments such as surgery and radiotherapy contain many inherent limitations with regards to incomplete and nonselective tumor ablation. Cold atomospheric plasma (CAP) is an ionized gas where the ion temperature is close to room temperature. It contains electrons, charged particles, radicals, various excited molecules, UV photons and transient electric fields. These various compositional elements have the potential to either enhance and promote cellular activity, or disrupt and destroy them. In particular, based on this unique composition, CAP could offer a minimally-invasive surgical approach allowing for specific cancer cell or tumor tissue removal without influencing healthy cells. Thus, the objective of this research is to investigate a novel CAP-based therapy for selectively bone metastatic breast cancer treatment. For this purpose, human metastatic breast cancer (BrCa) cells and bone marrow derived human mesenchymal stem cells (MSCs) were separately treated with CAP, and behavioral changes were evaluated after 1, 3, and 5 days of culture. With different treatment times, different BrCa and MSC cell responses were observed. Our results showed that BrCa cells were more sensitive to these CAP treatments than MSCs under plasma dose conditions tested. It demonstrated that CAP can selectively ablate metastatic BrCa cells in vitro without damaging healthy MSCs at the metastatic bone site. In addition, our study showed that CAP treatment can significantly inhibit the migration and invasion of BrCa cells. The results suggest the great potential of CAP for breast cancer therapy

The Strong Cell-based Hydrogen Peroxide Generation Triggered by Cold Atmospheric Plasma.

Hydrogen peroxide (H2O2) is an important signaling molecule in cancer cells. However, the significant secretion of H2O2 by cancer cells have been rarely observed. Cold atmospheric plasma (CAP) is a near room temperature ionized gas composed of neutral particles, charged particles, reactive species, and electrons. Here, we first demonstrated that breast cancer cells and pancreatic adenocarcinoma cells generated micromolar level H2O2 during just 1 min of direct CAP treatment on these cells. The cell-based H2O2 generation is affected by the medium volume, the cell confluence, as well as the discharge voltage. The application of cold atmospheric plasma (CAP) in cancer treatment has been intensively investigated over the past decade. Several cellular responses to CAP treatment have been observed including the consumption of the CAP-originated reactive species, the rise of intracellular reactive oxygen species, the damage on DNA and mitochondria, as well as the activation of apoptotic events. This is a new previously unknown cellular response to CAP, which provides a new prospective to understand the interaction between CAP and cells in vitro and in vivo. The short-lived reactive species in CAP may activate cells in vivo to generate long-lived reactive species such as H2O2, which may trigger immune attack on tumorous tissues via the H2O2-mediated lymphocyte activation

Cold atmospheric plasma modified electrospun scaffolds with embedded microspheres for improved cartilage regeneration

Articular cartilage is prone to degeneration and possesses extremely poor self-healing capacity due to inherent low cell density and the absence of a vasculature network. Tissue engineered cartilage scaffolds show promise for cartilage repair. However, there still remains a lack of ideal biomimetic tissue scaffolds which effectively stimulate cartilage regeneration with appropriate functional properties. Therefore, the objective of this study is to develop a novel biomimetic and bioactive electrospun cartilage substitute by integrating cold atmospheric plasma (CAP) treatment with sustained growth factor delivery microspheres. Specifically, CAP was applied to a poly(ε-caprolactone) electrospun scaffold with homogeneously distributed bioactive factors (transforming growth factor-β1 and bovine serum albumin) loaded poly(lactic-co-glycolic) acid microspheres. We have shown that CAP treatment renders electrospun scaffolds more hydrophilic thus facilitating vitronectin adsorption. More importantly, our results demonstrate, for the first time, CAP and microspheres can synergistically enhance stem cell growth as well as improve chondrogenic differentiation of human marrow-derived mesenchymal stem cells (such as increased glycosaminoglycan, type II collagen, and total collagen production). Furthermore, CAP can substantially enhance 3D cell infiltration (over two-fold increase in infiltration depth after 1 day of culture) in the scaffolds. By integrating CAP, sustained bioactive factor loaded microspheres, and electrospinning, we have fabricated a promising bioactive scaffold for cartilage regeneration

Integrating 3D Bioprinting and Nanomaterials for Complex Tissue Regeneration

Dr. Lijie Grace Zhang is an Associate Professor of Mechanical and Aerospace Engineering, as well as Biomedical Engineering and Medicine at George Washington University. Lecture Sponsors: School of Biological Sciences and College of Applied and Natural Science

Biomimetic nanocomposite hydrogels for cartilage regeneration

This chapter discusses current advancements in cartilage tissue regeneration with emphasis on the use of hydrogel-based nanocomposites for improved cartilage regeneration. Through the course of this chapter, we will begin with an introduction to cartilage regeneration focusing on material characteristics necessary for successful tissue regeneration. In addition, we will explore and evaluate varying scaffold fabrication strategies as well as the incorporation of biologics for in vitro evaluation and enhanced tissue formation. Lastly, a concise summary and future direction of cartilage tissue regeneration will be discussed with respect to clinical applications of hydrogel-based therapies

Nanotechnology in osteochondral regeneration

With the development of nanotechnology, a better understanding of the role of feature size (nano, micro, and macro) on cell and tissue behavior, and a focused effort of regenerative medicine and tissue engineering (TE) research on mimicking native tissue dimension and composition, staunch advancements have been made in the areas of tissue and organ regeneration. Through the course of this chapter, we will discuss and explore technological advancements at the nanoscale with emphasis in osteochondral (bone-cartilage) tissue regeneration. We will begin with a brief overview of the anatomy and physiology of osteochondral tissue then explore regenerative approaches for the disparate tissues (bone and cartilage) which comprise the osteochondral tissue unit. Next we will delve in to current strategies and techniques for the manufacture of gradient and stratified scaffolds for regeneration of the entire tissue unit with emphasis on synthetic and natural nanocomposite materials

Design of a novel 3D printed bioactive nanocomposite scaffold for improved osteochondral regeneration

Chronic and acute osteochondral defects as a result of osteoarthritis and trauma present a common and serious clinical problem due to the tissue’s inherent complexity and poor regenerative capacity. In addition, cells within the osteochondral tissue are in intimate contact with a 3D nanostructured extracellular matrix composed of numerous bioactive organic and inorganic components. As an emerging manufacturing technique, 3D printing offers great precision and control over the microarchitecture, shape, and composition of tissue scaffolds. Therefore, the objective of this study is to develop a biomimetic 3D printed nanocomposite scaffold with integrated differentiation cues for improved osteochondral tissue regeneration. Through the combination of novel nano-inks composed of organic and inorganic bioactive factors and advanced 3D printing, we have successfully fabricated a series of novel constructs which closely mimic the native 3D extracellular environment with hierarchical nanoroughness, microstructure, and spatiotemporal bioactive cues. Our results illustrate several key characteristics of the 3D printed nanocomposite scaffold to include improved mechanical properties as well as excellent cytocompatibility for enhanced human bone marrow-derived mesenchymal stem cell adhesion, proliferation, and osteochondral differentiation in vitro. The present work further illustrates the effectiveness of the scaffolds developed here as a promising and highly tunable platform for osteochondral tissue regeneration

Development of biomimetic and bioactive 3D nanocomposite scaffolds for osteochondral regeneration

Osteochondral tissue is composed of ordered and random biological nanostructures and can, in principal, be classified as a nanocomposite material. Thus, the objective of this research is to develop a novel biomimetic biphasic nanocomposite scaffold via a series of 3D fabricating techniques for osteochondral tissue regeneration. For this purpose, a highly porous Poly(caprolactone) (PCL) bone layer with bone morphogenetic protein-2 (BMP-2)-encapsulated Poly(dioxanone) (PDO) nanospheres and nanocrystalline hydroxyapatite was photocrosslinked to a Poly(ethylene glycol)-diacrylate (PEG-DA) cartilage layer containing transforming growth factor-β1 (TGF-β1)-encapsulated PLGA nanospheres. Novel tissue-specific growth factor-encapsulated nanospheres were efficiently fabricated via a wet co-axial electrospraying technique. Integration and porosity of the distinct layers was achieved via co-porogen leaching and ultraviolet (UV) photocrosslinking of water soluble poly(ethylene glycol) (PEG) and <150 um sodium chloride salt particles providing greater control over pore size and increased surface area. Our in vitro results showed significantly improved human bone marrow derived mesenchymal stem cells (hMSCs) adhesion and differentiation in bone and cartilage layers, respectively. In addition, we are working on developing a novel table top stereolithography (SL) apparatus for the manufacture of custom designed 3D biomimetic scaffolds with incorporated growth factor encapsulated nanospheres for osteochondral defect repair. Our early-stage SL development has illustrated good corroboration between computer-aided design (CAD) and manufactured constructs with controlled geometry. The ultimate goal of the novel tabletop SL system is the manufacture of patient-specific implantable 3D nanocomposite scaffolds for osteochondral defect repair. The current SL system developed in our lab allows for efficient photocrosslinking of two novel nanocomposite polymeric materials for the manufacture of three-dimensional (3D) osteochondral constructs with good spatiotemporal control of growth factor release in addition to exhibiting similar mechanical properties to that of the native tissues being addressed