Recently a protocol is established to obtain large quantities of human induced pluripotent stem cells (iPSC)-derived endothelial progenitors, called endothelial colony forming cells (ECFC), and of candidate smooth-muscle forming cells (SMFC). Here, the suitability for assembling in spheroids, and in larger 3D cell constructs is tested. iPSC-derived ECFC and SMFC are labeled with tdTomato and eGFP, respectively. Spheroids are formed in ultra-low adhesive wells, and their dynamic proprieties are studied by time-lapse microscopy, or by confocal microscopy. Spheroids are also tested for fusion ability either in the wells, or assembled on the Regenova 3D bioprinter which laces them in stainless steel micro-needles (the "Kenzan" method). It is found that both ECFC and SMFC formed spheroids in about 24 h. Fluorescence monitoring indicated a continuous compaction of ECFC spheroids, but stabilization in those prepared from SMFC. In mixed spheroids, the cell distribution changed continuously, with ECFC relocating to the core, and showing pre-vascular organization. All spheroids have the ability of in-well fusion, but only those containing SMFC are robust enough to sustain assembling in tubular structures. In these constructs a layered distribution of alpha smooth muscle actin-positive cells and extracellular matrix deposition is found. In conclusion, iPSC-derived vascular cell spheroids represent a promising new cellular material for scaffold-free biofabrication.
OBJECTIVES/GOALS: Our goal was to assess the ability of a 3D-Printed dual cover-core design alveolar ridge bone graft, to withstand the average maximum masticatory force of a healthy person. To this end, we characterized the materials, ran a finite element analysis (FEA) model, and validated it using a resin 3D-printed version tested under compression with strain gauges. METHODS/STUDY POPULATION: A tricalcium-phosphate/hydroxyapatite paste and mixed methacrylated alginate-gelatin were used for the core, and polycaprolactone for the cover. These were characterized using ASTM standards D695 and D638 for compression, tensile, and rheological testing. Then we converted cone CT-scan images of a mandibular alveolar ridge defect to an .stl file, and designed the cover and core in Meshmixer. The model was then imported into ANSYS 11.0, and a downward compression force of 500 N, the maximum masticatory force of a healthy adult, was applied on the graft and mandible’s top ridge. The different models included solid and porous covers and cores, as well as comparing screws on one or both sides of the cover, then validated by compressing a resin 3D-printed versions. RESULTS/ANTICIPATED RESULTS: The FEA model provided maximum displacements, Von Mises stress (VMS), and stress/strain values for each model. The highest maximum displacement was found on the solid covers with a combination of both buccal and lingual screws, at 0.162 mm. The lowest maximum displacement was found in the porous cover at 0.085 mm. All VMS values were below the tensile yield strength, meaning that the materials would not yield. The highest maximum stress was found on the porous cover at 13.52 MPa, the lowest was 1.06 MPa on the cover with no screws. The highest strain was found on the porous model at 0.010, which was 5.6x higher than the solid cover. The porous cover also showed less stress shielding, thus allowing a beneficial mechanical stimulation of the bone, and the lowest maximum displacement, possibly due to flexion through the pores. DISCUSSION/SIGNIFICANCE: Preliminary FEA models demonstrated that for the considered materials, a cover-core design of the mandibular implant would sustain the desired 500 N of force without yielding. The porous cover provides the most benefits, causing the least stress shielding and allowing diffusion of biological factors to support the osteoinductive role of the core.
Summary For the past two decades much research on selective photothermolysis of port wine stain vasculature has been devoted to optimizing laser parameters. Unfortunately, 60% of patients still respond suboptimally to laser therapy, despite significant innovations in treatment strategies and laser technology. Here we present a novel treatment approach based on combining selective photothermolysis with the administration of prothrombotic and/or anti-fibrinolytic pharmaceutical agents, with the aim of enhancing vaso-occlusion and post-treatment remodelling in difficult-to-target vessels. A hypercoagulable state of blood will instill laser-induced occlusive thrombosis in a wider array of vessel diameters at greater dermal depths, whereby larger vascular segments will ultimately undergo the chronic inflammatory processes that result in blood volume reduction, and thus lesional blanching. With thrombosis as a primary trigger for these inflammatory processes, we have extrapolated the thresh-old damage profile that is required for clinically relevant thrombus formation. Consequently, a recently proposed model of thrombus organization, in which recanalization is associated with endothelial progenitor cell-mediated neovasculogenesis, is elaborated in the framework of lesional blanching and juxtaposed to angiogenic reconstruction of affected dermal vasculature. Since neovasculogenesis and angiogenesis are regulated by the degree of vaso-occlusion and corollary drop in local oxygen tension, both can be manipulated by the administration of procoagulant pharmaceuticals. Lastly, in an effort to optimally balance selective photothermolysis with pharmacokinetics and clinical safety, the use of a gold nanoshell drug delivery system, in which the procoagulant drugs are encapsulated by a wavelength-modulated, gold-coated polymer matrix, is proposed. We have termed this modality site-specific pharmaco-laser therapy.
The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.
Apparently, ancient Greeks somehow learned that human liver could regenerate after damage. This information was connected in their intuition with their most powerful ability, that to produce fire. What is less often remembered is that in Hesiod’s version of Prometheus’ myth, after he has stolen the fire, Zeus also sent Pandora, the first woman, to all-male human beings in retaliation, counting on their greed as their greatest weakness. Despite Prometheus’ warning, human beings accepted Pandora’s jar as a gift from the gods, from which were released ‘evils, harsh pain and troublesome diseases which give men death’ (of note, again a medical connotation). Eventually, Pandora shut the lid of the jar, too late to contain the evils that escaped, and what remained inside was the hope. Thus, not only the evils were unlashed, but even hope became inaccessible: an allusion to the subjective effects of disease? It is largely assumed that the focus of the myth was on the punishment that the Gods inflicted on Prometheus, for his sacrilegious courage. If so, the only positive information, that on liver regeneration, would have remained at the periphery of the legend. But let’s try another reading of this myth, by bringing the surprisingly advanced medical knowledge to the centre: maybe these genial Greeks wanted to create a story grandiose enough, to be commensurate with the depth of their biological discovery, and have imagined a punishment ferocious enough to highlight by comparison the power of this organ repair. They came up to link the genealogical sin of the humankind – in their version – to this astonishing ability of at least a part of a living body to resist, be it momentarily, destruction and death. Thus the dream, if not the goal, of regenerative medicine was defined in this beautiful, ∼3000 years old myth. Nowadays, humankind seems to re-enact this tragedy (in the Antique meaning of the word), at another level: after taming the atomic fire, with new courage and ambition, but also with our perennial greed for goods, we now re-discover the power of tissue regeneration and that of the driven, medically justified, tissue repair or even de novo creation by ‘tissue engineering’. Thus, stem cells became the next revolution in biology. Most likely, soon there will be no organ spared from our attempt of intentional repair using these miraculous cells. But what are the stem cells anyway? What are the stem cells of the adult organism? What about cell plasticity and trans-differentiation? How do these cells naturally contribute to cell turnover and organ maintenance, and how do they change in aging? Could these cells be determined to a reversed differentiation (in fact, de-differentiation) pathway for resuming their journey to another phenotypic destination? How efficient is tissue engineering likely to be? And (thinking of Pandora), what are the bioethical implications of this regenerative medicine?…. Based on the often sad lessons of the past, there is little reason to believe that with these great gifts evils would not also come, and once released these evils could be ever brought back into Pandora’s jar. This new Progress in Regenerative Medicine Reviews series will address these and related questions in the following issues of the Journal. We are inviting the pioneers of this exploding field to share with our readers the essence of their exciting discoveries, to express their opinions about the current state and to forecast the future. By design, less emphasis will be placed on an exhaustive covering of the topics, already populated by expert reviews. Symbolically, the opening of the series is entrusted to Dr. Darwin Prockop, Director of the Institute of Regenerative Medicine in Temple, TX, member of National Academy of Sciences and of the Institute of Medicine, who is a pioneer in mesenchymal stem cells research. Besides a succinct discussion of the classical interpretations of their roles, he brings forth arguments for a new paradigm to explain the beneficial effects of these cells, namely that of formation of transient ‘quasi-niches’via inter-cellular communication of the more primitive, healthier cells with the injured tissue. In conclusion, accepting Pandora’s last gift, we hope that this new Reviews series is going to be not only a source of solid information about the latest progress in the field, but also a forum for courageous debate about the promises and perils of the Promethean tasks, which lay ahead of Regenerative Medicine. No conflicts of interests are associated with this Editorial.
The middle ear bones (‘ossicles’) may become severely damaged due to accidents or to diseases. In these situations, the most common current treatments include replacing them with cadaver-derived ossicles, using a metal (usually titanium) prosthesis, or introducing bridges made of biocompatible ceramics. Neither of these solutions is ideal, due to the difficulty in finding or producing shape-matching replacements. However, the advent of additive manufacturing applications to biomedical problems has created the possibility of 3D-printing anatomically correct, shape- and size-personalized ossicle prostheses. To demonstrate this concept, we generated and printed several models of ossicles, as solid, porous, or soft material structures. These models were first printed with a plottable calcium phosphate/hydroxyapatite paste by extrusion on a solid support or embedded in a Carbopol hydrogel bath, followed by temperature-induced hardening. We then also printed an ossicle model with this ceramic in a porous format, followed by loading and crosslinking an alginate hydrogel within the pores, which was validated by microCT imaging. Finally, ossicle models were printed using alginate as well as a cell-containing nanocellulose-based bioink, within the supporting hydrogel bath. In selected cases, the devised workflow and the printouts were tested for repeatability. In conclusion, we demonstrate that moving beyond simplistic geometric bridges to anatomically realistic constructs is possible by 3D printing with various biocompatible materials and hydrogels, thus opening the way towards the in vitro generation of personalized middle ear prostheses for implantation.
