Tissue Engineering Mid-Term Exam: Key Concepts & Questions

Tissue Engineering 580.442 / 580.642 MID-TERM EXAM – 1 (Fall 2021) This is a CLOSED BOOK exam. No notes, lecture slides, or other class materials should be available to you electronically or otherwise. You should be logged into the regular class zoom link with your video on while doing the exam. Question 1 – 9 points Question 4 – 13 points Question 2 – 12 points Question 5 – 11 points Question 3 – 15 points Total = 60 points 1. A flow chamber such as the one shown below may be used to quantify the strength of adhesion of cells to the underlying substrate. Cells are attached to the bottom surfaces and a fluid flows with a laminar profile through the chamber. a. Describe how this system might be used to measure the strength of cell adhesion to the substrate. ( Do NOT include any equations for this explanation – just describe the concepts with the key assumptions that might allow an experimenter to use flow to measure adhesion strength ). ( 4 points ) Since the flow applies pressure onto adherent cells, but not adherent cells, the strength of adhesion can be determined by the detachment tension for adherent cells from the surface of substrate when the fluid flows through the chamber. So if the fluid flow rate is known, we can7 calculate the strength of cell adhesion to the substrate.

b. Draw and label the main components of a focal adhesion to illustrate the extracellular matrix substrate, the binding proteins, at least 3 adaptor proteins, and the cytoskeletal proteins, connections to the nucleus. ( 5 points ).

2. Fibroblasts have been embedded in a 3D hydrogel substrate with binding sites as shown below. The hydrogel is tethered to posts at the top and bottom of the images but the long edges are not tethered. Over time, the hydrogel morphology changes as shown in the figure. a. Based on our discussions on how cells interact with their environments, sketch and describe what you expect the morphology and orientation of a random cell located in the center of the hydrogel to look like at each time point (i.e. Your answer should include 4 sketches of cells at Days 0, 7, 14, & 21 highlighting any changes in morphology that occur over time). Explain the reasoning behind your response. ( 8 points ).

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b. Copy your sketch for the cell alignment at Day 21 only. In this sketch, represent the alignment of the stress fibers in the cell. On 2 or 3 of these stress fibers, add arrows indicating what direction the stress (tension) in the fiber is acting. ( 2 points ) c. Sketch for Days 0 and 21 how would your might change if there were no binding sites in the gel to which the cells could attach ( 2 points )?

3. In the study by Kang et al, published in 2016, A 3D Bio-printing System to Produce Human Scale Tissue Constructs with Structural Integrity , the authors demonstrated that they could print large bone, cartilage, and skeletal muscle tissues. a. The bio-printer was used to print Pluronic 127, polycaprolactone, and cell-laden hydrogels. What was the function of each of these components? ( 3 points )? Pluronic sacrificial material to create channels, so it can be washed out after create space for diffusion. Channels make it possible for cells to receive oxygen and nutrients via diffusion. PCL: Can embed growth factors and bioactive agents promotes healing and it’s biodegradable, will be degraded in the body after implantation. Cell-laden hydrogel: Hydrogels can be engineered to mimic the extracellular matrix (ECM) of bone tissue. T hey can hold and protect cells, ensuring their viability during the printing process and in the host tissue. This is crucial for seeding the 3D-printed implant with the patient's own cells or with cells that promote bone regeneration. b. The hydrogel bio-ink was comprised of gelatin, fibrinogen, hyaluronic acid (HA), and glycerol. What was the role of each of these ( 2 points )? c. For that study out the chart below including at least one item in each cell of the table unless otherwise specificied ( 10 points ): Cell Type Inductive factor in medium In vitro Assessments In vivo Assessments

Bone  Osteocytes  Beta glycol phosphate  Ascorbic acid  Alizarin Red  microCT  vWF Cartilage  Chondrocytes  l-ascorbic acid  dexamethasone  Safranin O  Collagen II  MRI  Immunohistochemica l Skeletal Muscle  myocyt es  insulin  MHC  Desmin  MHC  Bioreactor for functional test 4. One strategy to facilitate rapid perfusion of engineered tissues with blood following implantation is to ‘pre-vascularize’ them using the strategy outlined below. In this schematic, the blue dots represent endothelial cells (1). During in vitro culture, the endothelial cells form a primitive vascular network (2). Upon in vivo implantation, vasculature (red lines) from the surrounding host tissues grows into the engineered graft and anastomoses with the pre-formed vasculature (3) allowing blood to flow through the vessels (4). a. What is the name of the process via which endothelial cells (such as human umbilical vein endothelial cells (HUVECs)) morph from individual cells to the primitive vascular network? ( 1 point ) vasculogensis b. In these cultures, an additional cell population (e.g. human mesenchymal stem cells (hMSCs)) is often included to act as ‘ pericytes’ . What is the role of pericytes/pericyte-like cells in these vascular structures? ( 1 point )

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c. Describe the homotypic interactions (between HUVECs) and heterotypic interactions (between HUVECS and hMSCs) that enable them to form stable capillary-like networks with perfusable lumens. In your description, please include the growth factors and molecular interactions that we discussed in class ( 7 points ). d. After implanting the graft in vivo, it would be important to demonstrate the ‘functionality’ of the engineered vascular networks. Describe an experimental approach you could use to demonstrate that engineered vasculature became functionally anastomosed with host vessels ( 4 points )?

5. New bio-printing techniques are being developed to directly “3D-print” composite tissue grafts such as liver or heart tissues. a. What are “ functional sub-units ”? Why have they been used for some 3D- bioprinting strategies? (3 points) b. Please describe 3 general properties of bio-inks that need to be controlled to successfully enable bio-printing of cells and state why they are important. (3 points) c. In the case study (Noor et al, 2019), what was the composition of the bio-ink and the rationale for using this bioink? ( 2 points ) d. How was gelation of the bioink (in Noor et al) effected? ( 1 point )

e. The authors used a sacrificial gelatin layer loaded with endothelial cells. However, when the gelatin degraded, the endothelial cells remain attached to the omental layer. Speculate on why the gelatin cells do not wash away? ( 2 points )

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