Three-dimensional photolithographic micropatterning: a novel tool to probe the complexities of cell migration

In order to independently study the numerous variables that influence cell movement, it will be necessary to employ novel tools and materials that allow for exquisite control of the cellular microenvironment. In this work, we have applied advanced 3D micropatterning technology, known as two-photon laser scanning lithography (TP-LSL), to poly(ethylene glycol) (PEG) hydrogels modified with bioactive peptides in order to fabricate precisely designed microenvironments to guide and quantitatively investigate cell migration. Specifically, TP-LSL was used to fabricate cell adhesive PEGRGDS micropatterns on the surface of non-degradable PEG-based hydrogels (2D) and in the interior of proteolytically degradable PEG-based hydrogels (3D). HT1080 cell migration was guided down these adhesive micropatterns in both 2D and 3D, as observed via time-lapse microscopy. Differences in cell speed, cell persistence, and cell shape were observed based on variation of adhesive ligand, hydrogel composition, and patterned area for both 2D and 3D migration. Results indicated that HT1080s migrate faster and with lower persistence on 2D surfaces, while HT1080s migrating in 3D were smaller and more elongated. Further, cell migration was shown to have a biphasic dependence on PEG-RGDS concentration and cells moving within PEG-RGDS micropatterns were seen to move faster and with more persistence over time. Importantly, the work presented here begins to elucidate the multiple complex factors involved in cell migration, with typical confounding factors being independently controlled. The development of this unique platform will allow researchers to probe how cells behave within increasingly complex 3D microenvironments that begin to mimic specifically chosen aspects of the in vivo landscape

Promotion and control of angiogenic activity in poly(ethylene glycol) diacrylate hydrogels for tissue engineering applications

Promotion and control of angiogenic activity have been achieved by covalently linking an angiogenic-signaling protein or peptide to poly(ethylene glycol) diacrylate hydrogels, thereby yielding a system which can be used to develop vascularized, functional engineered tissues. The field of tissue engineering has the capability of providing greatly needed biological organs and tissues for transplantation therapy. In living tissues of physiologically relevant size, metabolically active cells far from a nutrient source undergo necrosis when starved of oxygen and vital nutrients, but the incorporation of microvasculature would allow the production of functional tissues larger than 200 µm, the diffusion limit of oxygen through tissue. By combining a supporting matrix material, signaling factors, and cells, an environment has been created to support the formation and control of endothelial tubes. Poly(ethylene glycol) (PEG)-based hydrogels, which are hydrophilic and resistant to protein adsorption and subsequent non-specific cell adhesion, were modified to contain cell-adhesive ligands and growth factors to support cell and tissue function. Human endothelial cells were cultured in these systems in vitro. Covalent immobilization of vascular endothelial growth factor (VEGF) was shown to promote endothelial cell angiogenic activity, including migration, cell-cell contact formation, and tubule formation, in PEG hydrogels in two and three dimensions. Furthermore, patterning the micron-scale spatial presentation of cell-adhesive ligands and VEGF controlled and accelerated tubule formation. Endothelial tubules formed on restricted patterns less than 70 µm wide showed increased expression of angiogenic receptors VEGFR1, VEGFR2, and EphA7, in addition to increased production and secretion of laminin, a tubule-associated extracellular matrix protein. The incorporation of QK, a synthetic angiogenic peptide, was also shown to promote endothelial cell proliferation and tubulogenesis in two and three dimensions in vitro at levels comparable to those achieved through signaling by VEGF. This work improves upon previous research on angiogenic growth factor release from tissue engineering matrices by showing that localized, covalently-bound signaling can promote angiogenesis, thereby providing an engineered, predictable response in a local environment without systemic effects. Additionally, these findings provide a valuable method to incorporate capillary networks throughout tissue engineering matrices to support the continued development of functional tissue-engineered products for clinical use

Multimodality Imaging Evaluation of an Uncommon Entity: Esophageal Heterotopic Pancreas

A 25-year-old male was referred to the Radiology Department with new onset of right upper quadrant and epigastric abdominal pain. He had no past medical or surgical history. Physical exam was unremarkable. The patient underwent computed tomography (CT), fluoroscopic upper gastrointestinal (GI) evaluation, endoscopic ultrasound (EUS), and positron emission tomography (PET) evaluation, revealing the presence of a heterogeneous esophageal mass. In light of imaging findings and clinical workup, the patient was ultimately referred for thorascopic surgery. Surgical findings and histology confirmed the diagnosis of esophageal heterotopic pancreas

Micron-Scale Spatially Patterned, Covalently Immobilized Vascular Endothelial Growth Factor on Hydrogels Accelerates Endothelial Tubulogenesis and Increases Cellular Angiogenic Responses

Spontaneous formation of endothelial tubules was restricted to patterned micron-scale regions presenting cell adhesion ligands and angiogenic signaling protein on poly(ethylene glycol) hydrogels. Arginine-glycine-aspartic acid-serine (RGDS), an integrin ligand, and vascular endothelial growth factor (VEGF), a rate-limiting signaling protein involved in angiogenesis, were covalently bound through photopolymerization via laser scanning lithography to the surface of poly(ethylene glycol) hydrogels in patterned micron-scale regions. Endothelial cells cultured in this restricted environment underwent accelerated tubulogenesis, forming endothelial tubes within 2 days, whereas cells cultured on larger patterned areas remained spread and did not form tubules by day 2. Tubules formed in 2 days on RGDS and VEGF patterns were observed to possess lumens; however, tubule-like structures formed in 2 days on RGDS-only control patterns did not have observable lumens. Additionally, tubules that formed on restricted areas of RGDS and VEGF expressed more VEGF receptor 1, VEGF receptor 2, and ephA7 surface markers, in addition to higher expression of laminin, than cells remaining spread on wide patterned lines. This work reports spatial control and acceleration of endothelial tubule formation using biocompatible hydrogel materials to allow the formation of highly organized vascularized tissues