Enhanced rheological behaviors of alginate hydrogels with carrageenan for extrusion-based bioprinting.

Three-dimensional (3D) bioprinting using biocompatible materials is widely used in the field of tissue engineering and regenerative medicine. However, precise printing of 3D structures is challenging due to weak and uncontrollable mechanical properties of various hydrogels, thus limiting their potential in preclinical and clinical applications. In this study, our goal is to demonstrate the feasibility of precise fabrication of alginate/carrageenan composite scaffolds using extrusion-based 3D bioprinting. At first, the proper concentration of crosslinking agents was determined by the assessment of shear modulus of alginate-based hydrogels. Moreover, alginate/carrageenan composite hydrogels were prepared with different concentrations of carrageenan and used to measure their rheological properties. Based on the assessed viscosities and shear moduli of alginate and alginate/carrageenan hydrogels, printing resolutions in different printing parameters were simulated and presented in the printability maps. In addition, alginate and alginate/carrageenan scaffolds were bioprinted with various printing parameters and used to compare their printability with the simulated results. Also, 3D deposition of both alginate and alginate/carrageenan hydrogels were assessed and compared with each other by continuous monitoring of shape fidelity in 3D structures in ten layers and similar printing resolution. Finally, the cell viability of the 3D alginate/carrageenan composite scaffolds, printed using optimized printing parameters, was evaluated using live/dead staining and confocal fluorescence imaging. Thus, the results in the study show the potential uses of carrageenan for a prospective bioink with remarkable mechanical properties suitable for precise fabrication of 3D hydrogel scaffolds using bioprinting techniques.

In vivo photoacoustic monitoring using 700-nm region Raman source for targeting Prussian blue nanoparticles in mouse tumor model.

Photoacoustic imaging (PAI) is a noninvasive imaging tool to visualize optical absorbing contrast agents. Due to high ultrasonic resolution and superior optical sensitivity, PAI can be used to monitor nanoparticle-mediated cancer therapy. The current study synthesized Food and Drug Administration-approved Prussian blue (PB) in the form of nanoparticles (NPs) with the peak absorption at 712 nm for photoacoustically imaging tumor-bearing mouse models. To monitor PB NPs from the background tissue in vivo, we also developed a new 700-nm-region stimulated Raman scattering (SRS) source (pulse energy up to 200 nJ and repetition rate up to 50 kHz) and implemented optical-resolution photoacoustic microscopy (OR-PAM). The SRS-assisted OR-PAM system was able to monitor PB NPs in the tumor model with micrometer resolution. Due to strong light absorption at 712 nm, the developed SRS light yielded a two-fold higher contrast from PB NPs, in comparison with a 532-nm pumping source. The proposed laser source involved cost-effective and simple system implementation along with high compatibility with the fiber-based OR-PAM system. The study highlights the OR-PAM system in conjunction with the tunable-color SRS light source as a feasible tool to assist NP-mediated cancer therapy.

Hydroxyapatite Coated Iron Oxide Nanoparticles: A Promising Nanomaterial for Magnetic Hyperthermia Cancer Treatment.

Targeting cancer cells without injuring normal cells is the prime objective in treatment of cancer. In this present study, solvothermal and wet chemical precipitation techniques were employed to synthesize iron oxide (IO), hydroxyapatite (HAp), and hydroxyapatite coated iron oxide (IO-HAp) nanoparticles for magnetic hyperthermia mediated cancer therapy. The synthesized well dispersed spherical IO-HAp nanoparticles, magnetite, and apatite phases were confirmed by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and Field emission transmission electron microscopy (FETEM) with Energy Dispersive X-ray spectroscopy (EDS). The non-toxic behavior of synthesized IO-HAp nanoparticles was confirmed by cytotoxicity assay (Trypan blue and MTT assay). The synthesized nanoparticles revealed a remarkable magnetic saturation of 83.2 emu/g for IO and 40.6 emu/g for IO-HAp nanoparticles in presence of 15,000 Oe (1.5 T) magnetic field at room temperature (300 K). The magnetic hyperthermia study that was performed with IO-HAp nanoparticles showed an excellent hyperthermia effect (SAR value 85 W/g) over MG-63 osteosarcoma cells. The in vitro hyperthermia temperature (~45 °C) was reached within 3 min, which shows a very high efficiency and kills nearly all of the experimental MG-63 osteosarcoma cells within 30 min exposure. These results could potentially open new perceptions for biomaterials that are aimed for anti-cancer therapies based on magnetic hyperthermia.

Effect of multiple-sweeping on ablation performance during ex vivo laser nephrectomy.

BACKGROUND AND OBJECTIVE: Fiber-assisted laser surgery has been employed as a minimally invasive method in various medical fields. In spite of multiple sweeping on tissue during laser treatments, the rate of tissue removal gradually decreases and eventually leads to longer irradiation times as well as deeper thermal injury. The objective of the current study was to quantitatively investigate the effect of multiple fiber sweeps on ablation performance during ex vivo 532-nm laser nephrectomy. MATERIALS AND METHODS: Porcine kidney tissue was used to evaluate variations in tissue ablation and coagulative necrosis after pre- and multiple-sweeping with a 532 nm wavelength at various fiber speeds (2, 4, and 6 mm/second). The distance between a fiber tip and tissue surface was initially set at 1.5 mm, and no further distance change was performed. Double-integrating spheres in conjunction with an adding-doubling method were employed to measure variations in optical properties of the tested tissue. The extent of ablation and coagulation was quantified to identify the role of multiple-sweeping at various fiber conditions. RESULTS: Optical property measurements showed a 30% decrease in light absorption but a more than threefold increase in light scattering after irreversible thermal denaturation. Pre-sweeping yielded insignificant effects on tissue coagulation due to almost consistent coagulation depths with numbers of pre-sweeps. Ablation depths increased with more numbers of fiber sweeps and slower fiber speeds whereas coagulation depths thickened primarily with the slower speeds. Multiple-sweeping induced saturation in ablation volume with the increasing numbers of multiple-sweeps irrespective of the fiber speed. CONCLUSION: A combination of coagulation barriers, spatial distribution of power, and temporal interplay of optical energy could attribute to continuously lessen the amount of the ablated tissue with the multiple sweeps. Optical power modulation with varying fiber conditions (speed and distance) will be examined to optimize surgical parameters and to sustain the equivalent ablation performance of the first sweep with the multiple sweeping for laser nephrectomy. Lasers Surg. Med. 48:616-623, 2016. © 2016 Wiley Periodicals, Inc.

Intravascular ultrasonic-photoacoustic (IVUP) endoscope with 2.2-mm diameter catheter for medical imaging.

Intravascular ultrasound (IVUS) imaging is extremely important for detection and characterization of high-risk atherosclerotic plaques as well as gastrointestinal diseases. Recently, intravascular photoacoustic (IVPA) imaging has been used to differentiate the composition of biological tissues with high optical contrast and ultrasonic resolution. The combination of these imaging techniques could provide morphological information and molecular screening to characterize abnormal tissues, which would help physicians to ensure vital therapeutic value and prognostic significance for patients before commencing therapy. In this study, integration of a high-frequency IVUS imaging catheter (45MHz, single-element, unfocused, 0.7mm in diameter) with a multi-mode optical fiber (0.6mm in core diameter, 0.22 NA), an integrated intravascular ultrasonic-photoacoustic (IVUP) imaging catheter, was developed to provide spatial and functional information on light distribution in a turbid sample. Simultaneously, IVUS imaging was co-registered to IVPA imaging to construct 3D volumetric sample images. In a phantom study, a polyvinyl alcohol (PVA) tissue-mimicking arterial vessel phantom with indocyanine green (ICG) and methylene blue (MB) inclusion was used to demonstrate the feasibility of mapping the biological dyes, which are used in cardiovascular and cancer diagnostics. For the ex vivo study, an excised sample of pig intestine with ICG was utilized to target the biomarkers present in the gastrointestinal tumors or the atherosclerotic plaques with the proposed hybrid technique. The results indicated that IVUP endoscope with the 2.2-mm diameter catheter could be a useful tool for medical imaging.

Combined ultrasound and photoacoustic imaging to noninvasively assess burn injury and selectively monitor a regenerative tissue-engineered construct.

Current biomedical imaging tools have limitations in accurate assessment of the severity of open and deep burn wounds involving excess bleeding and severe tissue damage. Furthermore, sophisticated imaging techniques are needed for advanced therapeutic approaches such as noninvasive monitoring of stem cells seeded and applied in a biomedical 3D scaffold to enhance wound repair. This work introduces a novel application of combined ultrasound (US) and photoacoustic (PA) imaging to assess both burn injury and skin tissue regeneration. Tissue structural damage and bleeding throughout the epidermis and dermis till the subcutaneous skin layer were imaged noninvasively by US/PA imaging. Gold nanoparticle-labeled adipose-derived stem cells (ASCs) within a PEGylated fibrin 3D gel were implanted in a rat model of cutaneous burn injury. ASCs were successfully tracked till 2 weeks and were distinguished from host tissue components (e.g., epidermis, fat, and blood vessels) through spectroscopic PA imaging. The structure and function of blood vessels (vessel density and perfusion) in the wound bed undergoing skin tissue regeneration were monitored both qualitatively and semi-quantitatively by the developed imaging approach. Imaging-based analysis demonstrated ASC localization in the top layer of skin and a higher density of regenerating blood vessels in the treated groups. This was corroborated with histological analysis showing localization of fluorescently labeled ASCs and smooth muscle alpha actin-positive blood vessels. Overall, the US/PA imaging-based strategy coupled with gold nanoparticles has a great potential for stem cell therapies and tissue engineering due to its noninvasiveness, safety, selectivity, and ability to provide long-term monitoring.

Evaluation of gold nanotracers to track adipose-derived stem cells in a PEGylated fibrin gel for dermal tissue engineering applications.

Evaluating the regenerative capacity of a tissue-engineered device in a noninvasive and synchronous manner is critical to determining the mechanisms for success in clinical applications. In particular, directly tracking implanted cells in a three-dimensional (3D) scaffold is desirable in that it enables the monitoring of cellular activity in a specific and localized manner. The authors' group has previously demonstrated that the PEGylation of fibrin results in a 3D scaffold that supports morphologic and phenotypic changes in mesenchymal stem cells that may be advantageous in wound healing applications. Recently, the authors have evaluated adipose-derived stem cells (ASCs) as a mesenchymal cell source to regenerate skin and blood vessels due to their potential for proliferation, differentiation, and production of growth factors. However, tracking and monitoring ASCs in a 3D scaffold, such as a PEGylated fibrin gel, have not yet been fully investigated. In the current paper, nanoscale gold spheres (20 nm) as cell tracers for ASCs cultured in a PEGylated fibrin gel were evaluated. An advanced dual-imaging modality combining ultrasound and photoacoustic imaging was utilized to monitor rat ASCs over time. The ASCs took up gold nanotracers and could be detected up to day 16 with high sensitivity using photoacoustic imaging. There were no detrimental effects on ASC morphology, network formation, proliferation, and protein expression/secretion (ie, smooth muscle α-actin, vascular endothelial growth factor, matrix metalloproteinase-2, and matrix metalloproteinase-9) associated with gold nanotracers. Therefore, utilization of gold nanotracers can be an effective strategy to monitor the regenerative process of a stem cell source in a 3D gel for vascular and dermal tissue engineering applications.

Nonlinear photoacoustic signal increase from endocytosis of gold nanoparticles.

Nonlinear photoacoustic effects, rarely seen in biomedical photoacoustic imaging of tissues, can manifest themselves strongly when plasmonic nanoparticles are used as imaging contrast agents. Specifically, nonlinear behavior of photoacoustic signal with modest laser fluences can occur when nanoparticles undergo cellular endocytosis and aggregation leading to thermal coupling and subsequent localized temperature enhancement. Our study demonstrated this effect using in vitro tissue models containing cells. While the photoacoustic signal amplitude was linearly proportional to the cell/nanoparticle concentration, the photoacoustic signal increased nonlinearly as the laser fluence increased. Our results, therefore, suggest that the nonlinear effects can be exploited in molecular/cellular photoacoustic imaging.

In vivo ultrasound and photoacoustic monitoring of mesenchymal stem cells labeled with gold nanotracers.

Longitudinal monitoring of cells is required in order to understand the role of delivered stem cells in therapeutic neovascularization. However, there is not an imaging technique that is capable of quantitative, longitudinal assessment of stem cell behaviors with high spatial resolution and sufficient penetration depth. In this study, in vivo and in vitro experiments were performed to demonstrate the efficacy of ultrasound-guided photoacoustic (US/PA) imaging to monitor mesenchymal stem cells (MSCs) labeled with gold nanotracers (Au NTs). The Au NT labeled MSCs, injected intramuscularly in the lower limb of the Lewis rat, were detected and spatially resolved. Furthermore, our quantitative in vitro cell studies indicate that US/PA imaging is capable of high detection sensitivity (1×104 cells/mL) of the Au NT labeled MSCs. Finally, Au NT labeled MSCs captured in the PEGylated fibrin gel system were imaged in vivo, as well as in vitro, over a one week time period, suggesting that longitudinal cell tracking using US/PA imaging is possible. Overall, Au NT labeling of MSCs and US/PA imaging can be an alternative approach in stem cell imaging capable of noninvasive, sensitive, quantitative, longitudinal assessment of stem cell behaviors with high spatial and temporal resolutions at sufficient depths.

Function of mesenchymal stem cells following loading of gold nanotracers.

BACKGROUND: Stem cells can differentiate into multiple cell types, and therefore can be used for cellular therapies, including tissue repair. However, the participation of stem cells in tissue repair and neovascularization is not well understood. Therefore, implementing a noninvasive, long-term imaging technique to track stem cells in vivo is needed to obtain a better understanding of the wound healing response. Generally, we are interested in developing an imaging approach to track mesenchymal stem cells (MSCs) in vivo after delivery via a polyethylene glycol modified fibrin matrix (PEGylated fibrin matrix) using MSCs loaded with gold nanoparticles as nanotracers. The objective of the current study was to assess the effects of loading MSCs with gold nanoparticles on cellular function. METHODS: In this study, we utilized various gold nanoparticle formulations by varying size and surface coatings and assessed the efficiency of cell labeling using darkfield microscopy. We hypothesized that loading cells with gold nanotracers would not significantly alter cell function due to the inert and biocompatible characteristics of gold. The effect of nanoparticle loading on cell viability and cytotoxicity was analyzed using a LIVE/DEAD stain and an MTT assay. The ability of MSCs to differentiate into adipocytes and osteocytes after nanoparticle loading was also examined. In addition, nanoparticle loading and retention over time was assessed using inductively coupled plasma mass spectrometry (ICP-MS). CONCLUSION: Our results demonstrate that loading MSCs with gold nanotracers does not alter cell function and, based on the ICP-MS results, long-term imaging and tracking of MSCs is feasible. These findings strengthen the possibility of imaging MSCs in vivo, such as with optical or photoacoustic imaging, to understand better the participation and role of MSCs in neovascularization.